Cell based signal generation

ABSTRACT

The present invention makes available a rapid, reproducible, robust assay system for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular protein, e.g., a receptor or ion channel. The subject assay enables rapid screening of large numbers of compounds to identify those which act as an agonist or antagonist to the bioactivity of the cellular protein. In particular, the assay of the invention makes use of a cell that harbors a protein that is responsive to a cellular signal transduction pathway. The protein is operatively linked to a polypeptide which causes a detectable signal to be generated upon stimulation of the pathway, e.g., when a compound interacts with and modulates the activity of a cellular receptor or ion channel of the cell. Thus, the cell provides a signal generation means comprising a novel fusion protein the expression of which is independent of stimulation/activation of the signal transduction pathway, but the activity of which is responsive to the signal transduction pathway.

This application is a continuation of U.S. application Ser. No.09/241,888, filed Feb. 1, 1999, now issued as U.S. Pat. No. 6,504,008.

BACKGROUND OF THE INVENTION

The identification of biological activity in new molecules hashistorically been accomplished through the use of in vitro assays orwhole animals. Intact biological entities, either cells or wholeorganisms, have been used to screen for anti-bacterial, anti-fungal,anti-parasitic and anti-viral agents in vitro. Cultured mammalian cellshave also been used in screens designed to detect potential therapeuticcompounds. A variety of bioassay endpoints have been exploited in cellscreens including the stimulation of growth or differentiation of cells,changes in cell motility, the production of particular metabolites, theexpression of specific proteins within cells, altered protein function,and altered conductance properties. Cytotoxic compounds used in cancerchemotherapy have been identified through their ability to inhibit thegrowth of tumor cells in vitro and in vivo. In addition to cultures ofdispersed cells, whole tissues have served in bioassays, as in thosebased on the contractility of muscle.

In vitro testing is a preferred methodology in that it permits thedesign of high-throughput screens: small quantities of large numbers ofcompounds can be tested in a short period of time and at low expense.Optimally, animals are reserved for the latter stages of compoundevaluation and are not used in the discovery phase, inasmuch as the useof whole animals is labor-intensive and extremely expensive.

The search for agonists and antagonists of cellular receptors has beenan intense area of research aimed at drug discovery because of theelegant specificity of these molecular targets. Drug screening has beencarried out using whole cells expressing functional receptors and,recently, binding assays employing membrane fractions or purifiedreceptors have been designed to screen compound libraries forcompetitive ligands.

The heterologous expression of recombinant mammalian G protein-coupledreceptors in mammalian cells which do not normally express thosereceptors has been described as a means of studying receptor functionfor the purpose of identifying agonists and antagonists of thosereceptors. For example, the human muscarinic receptor (HM1) has beenfunctionally expressed in mouse cells (Harpold et al. U.S. Pat. No.5,401,629). The rat V1b vasopressin receptor has been found to stimulatephosphotidylinositol hydrolysis and intracellular Ca²⁺ mobilization inChinese hamster ovary cells upon agonist stimulation (Lolait et al.(1995) Proc. Natl. Acad Sci. USA 92:6783–6787). These types of ectopicexpression studies have enabled researchers to study receptor signalingmechanisms and to perform mutagenesis studies which have been useful inidentifying portions of receptors that are critical for ligand bindingor signal transduction.

Experiments have also been undertaken to express functional Gprotein-coupled receptors in yeast cells. For example, U.S. Pat. No.5,482,835 to King et al. describes a transformed yeast cell which isincapable of producing a yeast G protein α subunit, but which has beenengineered to produce both a mammalian G protein α-subunit and amammalian receptor which is “coupled to” (ie., interacts with) theaforementioned mammalian G protein α-subunit. Specifically, U.S. Pat.No. 5,482,835 reports expression of the human beta-2 adrenergic receptor(β2AR), a seven transmembrane receptor (STR), in yeast, under control ofthe GAL1 promoter, with the β2AR gene modified by replacing the first 63base pairs of coding sequence with 11 base pairs of noncoding and 42base pairs of coding sequence from the STE2 gene. (STE2 encodes theyeast α-factor receptor.) It was found that the modified β2AR wasfunctionally integrated into the membrane, as shown by studies of theability of isolated membranes to interact properly with various knownagonists and antagonists of β2AR. The ligand binding affinity foryeast-expressed β2AR was said to be nearly identical to that observedfor naturally produced β2AR.

U.S. Pat. No. 5,482,835 also describes co-expression of a rat G proteinα-subunit in yeast strain 8C, which lacks the cognate yeast protein.Ligand binding resulted in G protein-mediated signal transduction. U.S.Pat. No. 5,482,835 further teaches that these cells may be used inscreening compounds for the ability to affect the rate of dissociationof Gα from Gβγ in a cell. For this purpose, the cell further contains apheromone-responsive promoter (e.g., BAR1 or FUS1), linked to anindicator gene (e.g. HIS3 or lacZ). The cells are placed in multi-titerplates, and different compounds are placed in each well. The coloniesare then scored for expression of the indicator gene.

U.S. Pat. No. 5,789,184 describes yeast cells engineered to express aheterologous kinase as a yeast pheromone system protein surrogate, and aheterologous polypeptide. The yeast cells are used in assays to screenfor peptides that modulate the activity of non-yeast surrogates.

Published PCT international application WO 98/13513 describes methodsfor identifying modulators of heterologous receptors expressed in yeast.Modulators are identified by detecting an alteration in a signalproduced by an endogenous yeast signaling pathway.

U.S. Pat. No. 4,833,080 discloses regulation of eukaryotic geneexpression using a two component chimeric fusion protein. The chimericfusion protein consists of a DNA binding domain and a transcriptionalactivation domain.

SUMMARY OF THE INVENTION

The present invention relates to a novel, rapid, reproducible, robustassay system for screening and identifying pharmaceutically effectivecompounds that specifically interact with and modulate the activity of acellular receptor or ion channel of a cell.

The assay of the invention makes use of a cell that harbors a proteinthat is responsive to a cellular signal transduction pathway. Theprotein is operatively linked to a polypeptide which causes a detectablesignal to be generated upon stimulation of the pathway, e.g., when acompound interacts with and modulates the activity of a cellularreceptor or ion channel of the cell. Thus, the cell provides a signalgeneration means comprising a novel fusion protein the expression ofwhich is independent of stimulation/activation of the signaltransduction pathway, but the activity of which is responsive to thesignal transduction pathway.

The present invention provides for the use of any type of cell in thesubject assays, whether prokaryotic or eukaryotic. In preferredembodiments, the cells of the present invention are eukaryotic. Incertain preferred embodiments the cells are mammalian cells. In otherpreferred embodiments the cells are yeast cells, with cells from thegenera Saccharomyces or Schizosaccharomyces being more preferred. Thehost cells can be derived from primary cells, or from transformed and/orimmortalized cell lines.

The subject assays provide a means for detecting the ability ofcompounds to modulate the signal transduction activity of the targetreceptor by scoring for up- or down-regulation of a detection signal.Signal transduction can be measured in a variety of ways, including butnot limited to, physical and biological methods, enzymatic methods, andtranscriptional activation of endogenous genes or reporter genes. Forexample, endogenous yeast second messenger generation (e.g., GTPhydrolysis, calcium mobilization, or phospholipid hydrolysis) orincreased transcription of an endogenous gene can be detected directly.Alternatively, the use of a reporter or indicator gene can provide aconvenient readout. By whatever means measured, a change (e.g., astatistically significant change) in the detection signal can be used tofacilitate isolation of those cells from the mixture which have receiveda signal via the target receptor, and thus can be used to identify novelcompounds which function as receptor agonists or antagonists.

In one embodiment of the present invention, the reagent cells expressthe receptor of interest endogenously. In other embodiments, the cellsare engineered to express a heterologous receptor protein. In either ofthese embodiments, it may be desirable to inactivate one or moreendogenous genes of the host cells. For example, certain preferredembodiments in which a heterologous receptor is provided utilize hostcells in which the gene for the homologous receptor has beeninactivated. Likewise, other proteins involved in transducing signalsfrom the target receptor can be inactivated, or complemented with anortholog or paralog from another organism, e.g., yeast G proteinsubunits can be complemented by mammalian G protein subunits in yeastcells also engineered to express a mammalian G protein coupled receptor.Other complementations include, for example, expression of heterologousMAP kinases or erk kinases, MEKs or MKKs (MAP kinase kinases), MEKKs(MEK kinases), PAKs (p21-activated kinases, e.g., Ste 20), ras and thelike.

In one embodiment, the assay of the present invention can be used toscreen compounds, e.g., small molecules, which are exogenously added tocells in order to identify potential receptor effector compounds. Inanother embodiment the subject assays enable rapid screening of largenumbers of polypeptides in a library expressed in the cell in order toidentify those polypeptides which agonize or antagonize receptorbioactivity, creating an autocrine system. The autocrine assay ischaracterized by the use of a library of recombinant cells, each cell ofwhich includes a target receptor protein whose signal transductionactivity can be modulated by interaction with an extracellular signal,the transduction activity being able to generate a detectable signal,and an expressible recombinant gene encoding an exogenous testpolypeptide from a polypeptide library. By the use of a gene library,the mixture of cells collectively expresses a population of testpolypeptides. In preferred embodiments, the polypeptide library includesat least 10³ different polypeptides, though more preferably at least10⁵, 10 ⁶, or 10⁷ different (variegated) polypeptides. The polypeptidelibrary can be generated as a random peptide library, as a semi-randompeptide library (e.g., based on combinatorial mutagenesis of a knownligand), or as a cDNA library.

In another embodiment of the assay, if a test compound does not appearto directly induce the activity of the receptor protein, the assay maybe repeated and modified by the introduction of a step in which the cellis first contacted with a known activator of the target receptor toinduce the signal transduction pathways from the receptor. Thus, a testcompound can be assayed for its ability to antagonize, e.g., inhibit orblock the activity of the activator. Alternatively, the assay can scorefor compounds which potentiate the induction response generated bytreatment of the cell with a known activator.

A particularly advantageous feature of the invention is a recombinantyeast cell that harbors a yeast protein that is sensitive/responsive toa signaling pathway of the yeast cell, and that is operatively linked toa polypeptide, such that the polypeptide causes a detectable signal tobe generated when the pathway is stimulated.

Accordingly, in one aspect, the invention features a recombinant yeastcell comprising a yeast protein operatively linked to a polypeptide,wherein the yeast protein is responsive to a signal transduction pathwayof the cell, and the activity of the yeast protein is modulated directlyor indirectly upon stimulation of the pathway, and wherein uponmodulation of the yeast protein, the polypeptide causes a detectablesignal to be generated.

In one embodiment, the signal transduction pathway is a yeast pheromoneresponse pathway.

In another embodiment, the recombinant yeast cell further comprises agene which produces a detectable protein. In a preferred embodiment, thegene that produces the detectable protein is selected from the groupconsisting of ADE1, ADE2, ADE3, ADE4, ADE5, ADE7, ADE8, ASP3, ARG1,ARG3, ARG4, ARG5, ARG6, ARG8, ARO2, ARO7, BAR1, CAT, CHO1, CYS3, GAL1,GAL7, GAL10, GFP, HIS1, HIS3, HIS4, HIS5, HOM3, HOM6, ILV1, ILV2, ILV5,INO1, INO2, INO4, lacZ, LEU1, LEU2, LEU4, luciferase, LYS2, MAL, MEL,MET2, MET3, MET4, MET8, MET9, MET14, MET16, MET19, OLE1, PHO5, PRO1,PRO3, THR1, THR4, TRP1, TRP2, TRP3, TRP4, TRP5, URA1, URA2, URA3, URA4,URA5 and URA10. In another preferred embodiment, the gene is selectedfrom the group consisting of CAT, GAL1, GAL7, GAL10, GFP, HIS3, lacZ,luciferase, LEU2, MEL, PHO5, and URA3. In yet another embodiment, thegene is an endogenous gene at its natural location in the yeast cell,and the polypeptide causes the endogenous yeast gene to produce thedetectable protein. In a preferred embodiment, the natural location inthe yeast cell is the natural location of the endogenous gene in theyeast genome.

In one embodiment, the detectable protein is selected from the groupconsisting of α-galactosidase, β-galactosidase, alkaline phosphatase,horseradish peroxidase, exoglucanase, luciferase, Bar1, Pho5 acidphosphatase, green fluorescent protein, chitinase, and chloramphenicolacetyl transferase. In a preferred embodiment, the detectable protein isβ-galactosidase.

In another embodiment, the recombinant yeast cell further comprises aheterologous receptor that is functionally integrated into the signaltransduction pathway. In a preferred embodiment, the heterologousreceptor is expressed in the cell membrane of the yeast cell. In anotherpreferred embodiment, the heterologous receptor is selected from thegroup consisting of melatonin receptor 1a, galanin receptor 1,neurotensin receptor, adenosine receptor 2a, somatostatin receptor 2,and corticotropin releasing factor receptor 1. In yet another preferredembodiment, the heterologous receptor is melatonin receptor 1a.

In one embodiment, the activity of the yeast protein is directlymodulated upon stimulation of the signal transduction pathway. In apreferred embodiment, the yeast protein is a wild type yeast protein.

In another embodiment, the activity of the yeast protein is indirectlymodulated upon stimulation of the signal transduction pathway. In apreferred embodiment, the yeast protein is a mutant yeast protein, theactivity of which is modulated by a wild type yeast protein, and whereinthe activity of the wild type yeast protein is directly modulated uponstimulation of the signal transduction pathway. In another preferredembodiment, the mutant yeast protein is derived from the wild type yeastprotein.

In one embodiment, the yeast protein is selected from the groupconsisting of Fus3, Hog1, Kss1, Mpk1, Smk1, Bem1, Cdc24, Cdc42, Dig1,Dig2, Far1, Gpa1, Msg5, Ste4, Ste5, Ste7, Ste11, Ste12, Ste18, Ste20 andSst2. In preferred embodiments, the yeast protein is Fus3, Hog1, Kss1,Far1, Ste5 and Ste11.

In one embodiment, the polypeptide is a transcription factor. Thetranscription factor is selected from the group consisting of bacterial,viral and eukaryotic transcription factors. In a preferred embodiment,the transcription factor is a yeast transcription factor. The yeasttranscription is selected from the group consisting of Ste12, Gal4,Pho4, Gcn4, Hap1, Adr1, Ace2, Cup2, Swi5 and Bas1. In preferredembodiments, the yeast transcription factor is Ste12, Gal4, and Pho4.

In another embodiment, the polypeptide is a chimeric transcriptionfactor comprising a DNA binding domain (DBD) operatively linked to atranscriptional activation domain (AD). In one embodiment, the DNAbinding domain and transcriptional activation domain are derived fromthe same protein. In another embodiment, the DNA binding domain andtranscriptional activation domain are derived from different proteins.In a preferred embodiment, the DNA binding domain comprises apolypeptide sequence derived from a polypeptide selected from the groupconsisting of LexA, Gal4, Adr1, Ace2, Cup2, Bas1, Gcn4, Swi5, Pho4, Hap1and LacI, and the transcriptional activation domain comprises apolypeptide sequence derived from a polypeptide selected from the groupconsisting of B42, Gal4, Adr1, Ace2, Cup2, Bas1, Gcn4, Swi5, Pho4, Hap1,VP16, and Ste12.

In preferred embodiments, the DNA binding domain comprises a polypeptidesequence derived from LexA, from Gal4, and from Pho4. In other preferredembodiments, the transcriptional activation domain comprises apolypeptide sequence derived from B42, from VP16, from Gal4, or fromSte12.

In particularly preferred embodiments, the chimeric transcription factorcomprises: a LexA DNA binding domain operatively linked to a B42transcriptional activation domain; a Gal4 DNA binding domain operativelylinked to a B42 transcriptional activation domain; a Gal4 DNA bindingdomain operatively linked to a VP16 transcriptional activation domain;and a Gal4 DNA binding domain operatively linked to a Gal4transcriptional activation domain II.

In one embodiment, the recombinant yeast cell further comprises apromoter, wherein transcription of the gene that produces the detectableprotein is controlled by the promoter. Upon modulation of the activityof the yeast protein, the transcription factor activates transcriptionof the gene that produces the detectable protein. In a preferredembodiment, the promoter is selected from the group consisting of Gal1,Gal10, Mel and LexA operator. In another embodiment, the promoter isoperatively linked to an endogenous gene in its natural location in theyeast cell.

In another embodiment, the polypeptide comprises a protein that can bean enzyme, a protein required for cell viability, or an indicatormolecule. The protein is selected from the group consisting of Ade1,Ade2, Ade3, Ade4, Ade5, Ade7, Ade8, Asp3, Arg1, Arg3, Arg4, Arg5, Arg6,Arg8, Aro2, Aro7, Bar1, CAT, Cho1, Cys3, Gal1, Gal7, Gal10, GFP, His1,His3, His4, His5, Hom3, Hom6, Ilv1, Ilv2, Ilv5, Ino1, Ino2, Ino4, lacZ,Leu1, Leu2, Leu4, luciferase, Lys2, Mal, Mel, Met2, Met3, Met4, Met8,Met9, Met14, Met16, Met19, Ole1, Pho5, Pro1, Pro3, Thr1, Thr4, Trp1,Trp2, Trp3, Trp4, Trp5, Ura1, Ura2, Ura3, Ura4, Ura5, Ura10, Cdc25, Cyr1and Ras, or fragment thereof. In a preferred embodiment, the polypeptideis an enzyme. In another preferred embodiment, the enzyme is selectedfrom the group consisting of CAT, Gal1, lacZ, Mel, and Pho5. In anotherpreferred embodiment, the protein is a protein required for cellviability. In yet another preferred embodiment, the protein required forcell viability is selected from the group consisting of Gal1, His3,Leu2, Mel, Ura3, Cdc25, Cyr1 and Ras. In another embodiment, thepolypeptide is an indicator molecule. In a preferred embodiment, theindicator molecule is GFP.

In another embodiment, the yeast cell further comprises a heterologoustest polypeptide, wherein the heterologous test polypeptide istransported to a location allowing interaction with the extracellularregion of the heterologous receptor, and wherein the heterologous testpolypeptide is expressed at a sufficient level such that modulation ofthe signal transduction activity of the receptor by the heterologoustest polypeptide alters the detectable signal. In a preferredembodiment, the heterologous test polypeptide includes a signal sequencethat facilitates transport of the polypeptide to a location allowinginteraction with the extracellular region of the receptor.

In another aspect, the invention features a recombinant yeast cell asherein above described, except that a pheromone responsive yeast proteinis operatively linked to a chimeric transcription factor such that thechimeric transcription factor causes a detectable signal to be generatedupon stimulation of a yeast pheromone response pathway. Thus, theinvention is directed to a recombinant yeast cell comprising a yeastprotein operatively linked to a chimeric transcription factor, and agene which produces a detectable protein, wherein the yeast protein isresponsive to a yeast pheromone response pathway, and the activity ofthe yeast protein is modulated directly or indirectly upon stimulationof the pathway of the yeast cell, and wherein upon modulation of theyeast protein, the chimeric transcription factor causes a detectablesignal to be generated.

In one embodiment, the yeast cell further comprises a heterologousreceptor that is functionally integrated into the yeast pheromoneresponse pathway. In a preferred embodiment, the heterologous receptoris selected from the group consisting of melatonin receptor 1a, galaninreceptor 1, neurotensin receptor, adenosine receptor 2a, somatostatinreceptor 2, and corticotropin releasing factor receptor 1. In anotherpreferred embodiment, the heterologous receptor is melatonin receptor1a.

In a preferred embodiment, the yeast protein is wild type Fus3. Inanother preferred embodiment, the yeast protein is mutated Fus3. Themutated Fus3 may comprise an amino acid substitution at positions 42,180 or 182, or at positions 180 and 182, of the wild type Fus3 aminoacid sequence. Preferred substitutions at position 42 include arginine;at position 180, valine and glutamic acid; and at position 182,asparagine and valine. Preferred amino acid substitutions in the wildFus3 yeast protein are Lys42Arg, Thr180Val, Thr180Glu, Try182Val, andTry182Asp.

In another preferred embodiment, the chimeric transcription factorcomprises: a LexA DNA binding domain operatively linked to a B42transcriptional activation domain; a Gal4 DNA binding domain operativelylinked to a B42 transcriptional activation domain; a Gal4 DNA bindingdomain operatively linked to a VP16 transcriptional activation domain;and a Gal4 DNA binding domain operatively linked to a Gal4transcriptional activation domain II.

In yet another embodiment, the recombinant yeast cell further comprisesa promoter operatively linked to the gene, such that upon modulation ofthe activity of the yeast protein that is responsive to the yeastpheromone response pathway of the cell, the activity of the chimerictranscription factor is stimulated, thereby activating transcription ofthe gene that produces the detectable protein. In a preferredembodiment, the promoter comprises the minimal Gal promoter and LexAoperators, the gene is the lacZ gene, and the detectable protein isβ-galactosidase.

In another aspect, the invention features methods that use therecombinant yeast cells herein above described to identify compoundsthat modulate a receptor expressed by the recombinant yeast cells. Thus,the invention is directed to a method for identifying a test compoundthat modulates a receptor expressed by a recombinant yeast cellcomprising:

providing a recombinant yeast cell that expresses the receptor which isfunctionally integrated into a signal transduction pathway of the yeastcell, wherein the yeast cell comprises a yeast protein which isresponsive to the signal transduction pathway and which is operativelylinked to a polypeptide, and wherein the activity of said yeast proteinis modulated directly or indirectly upon stimulation of the pathway, andwherein upon modulation of the yeast protein, the polypeptide causes adetectable signal to be generated;

contacting the yeast cell with a test compound; and

detecting an alteration in the signal to thereby identify a compoundthat modulates the receptor.

The invention is also directed to a method for identifying a testcompound that modulates a receptor expressed by a recombinant yeast cellcomprising:

providing a recombinant yeast cell that expresses a receptor which isfunctionally integrated into a yeast pheromone response pathway, whereinthe yeast cell comprises a yeast protein which is responsive to thepheromone response pathway and which is operatively linked to a chimerictranscription factor, and a gene which produces a detectable protein,and wherein the activity of the yeast protein is modulated directly orindirectly upon stimulation of the pathway, and wherein upon modulationof the yeast protein, the chimeric transcription factor causes adetectable signal to be generated;

contacting the yeast cell with a test compound; and

detecting an alteration in the signal to thereby identify a compoundthat modulates the receptor.

In one embodiment of the methods of the invention, the test compound isderived from a peptide library. In another embodiment, the test compoundis derived from a library of non-peptidic compounds. In yet anotherembodiment, the test compound is derived from a library of testpolypeptides expressed by the cell.

In one embodiment, the step of detecting the alteration in the signalcomprises measuring the transcription of an endogenous gene or theactivity of an endogenous protein in the cell.

In another aspect, the invention features a chimeric nucleic acidconstruct comprising:

a first segment comprising a nucleotide sequence encoding a yeastprotein or fragment thereof which is responsive to a signal transductionpathway of a yeast cell, wherein the activity of the yeast protein orfragment thereof is modulated directly or indirectly upon stimulation ofa the signal transduction pathway; and

a second segment comprising a nucleotide sequence encoding a polypeptidethat causes a detectable signal to be generated upon modulation of theyeast protein.

In one embodiment, the signal transduction pathway is a yeast pheromonepathway. In another embodiment, the first segment encodes a yeastprotein selected from the group consisting of Fus3, Hog1, Kss1, Mpk1,Smk1, Bem1, Cdc24, Cdc42, Dig1, Dig2, Far1, Gpa1, Msg5, Ste4, Ste5,Ste7, Ste11, Ste12, Ste18, Ste20 and Sst2. In preferred embodiments, thefirst segment encodes Fus3, Far1, Ste5, Ste11, and Ste12.

In one embodiment, the second segment encodes a polypeptide comprising atranscription factor. In a preferred embodiment, the second segmentencodes a polypeptide selected from the group of yeast transcriptionfactors. These include, but are not limited to, Ste12, Gal4, Pho4, Gcn4,Hap1, Adr1, Ace2, Cup2, Swi5 and Bas1.

In another embodiment, the second segment encodes a chimerictranscription factor. The second segment comprises a third segmentcomprising a nucleotide sequence encoding a DNA binding domain, and afourth segment comprising a nucleotide sequence encoding atranscriptional activation domain. In one embodiment, the third andfourth segments are derived from the same gene. In another embodiment,the third and fourth segments are derived from different genes. Inpreferred embodiments, the third segment encodes a polypeptidecomprising: LexA DNA binding domain; or a Gal4 DNA binding domain. Inother preferred embodiments, the fourth segment encodes a polypeptidecomprising: a B42 transcriptional activation domain; or a Gal4transcriptional activation domain II.

In another embodiment, the second segment encodes a polypeptidecomprising a protein that can be an enzyme, a protein required for cellviability, or an indicator molecule. The protein is selected from thegroup consisting of Ade1, Ade2, Ade3, Ade4, Ade5, Ade7, Ade8, Asp3,Arg1, Arg3, Arg4, Arg5, Arg6, Arg8, Aro2, Aro7, Bar1, CAT, Cho1, Cys3,Gal1, Gal7, Gal10, GFP, His1, His3, His4, His5, Hom3, Hom6, Ilv1, Ilv2,Ilv5, Ino1, Ino2, Ino4, lacZ, Leu1, Leu2, Leu4, luciferase, Lys2, Mal,Mel, Met2, Met3, Met4, Met8, Met9, Met14, Met16, Met19, Ole1, Pho5,Pro1, Pro3, Thr1, Thr4, Trp1, Trp2, Trp3, Trp4, Trp5, Ura1, Ura2, Ura3,Ura4, Ura5, Ura10, Cdc25, Cyr1 and Ras, or fragment thereof In apreferred embodiment, the polypeptide is an enzyme. In another preferredembodiment, the enzyme is selected from the group consisting of CAT,Gal1, lacZ, Mel, and Pho5. In another preferred embodiment, the proteinis a protein required for cell viability. In yet another preferredembodiment, the protein required for cell viability is selected from thegroup consisting of Gal1, His3, Leu2, Mel, Ura3, Cdc25, Cyr1 and Ras. Inanother embodiment, the protein is an indicator molecule. In a preferredembodiment, the indicator molecule is GFP.

In another aspect, the invention features fusion proteins encoded by thechimeric nucleic acid constructs herein above described.

In one embodiment, the invention is directed to a fusion proteincomprising a yeast protein responsive to a signal transduction pathwayof a yeast cell operatively linked to a protein that is an enzyme, aprotein required for cell viability, or an indicator molecule. In apreferred embodiment, the fusion protein comprises Ste5 operativelylinked to Ras. In another preferred embodiment, the fusion proteincomprises Ste11 operatively linked to His3.

In another embodiment, the invention is directed to a fusion proteincomprising a yeast protein responsive to a signal transduction pathwayof a yeast cell operatively linked to a chimeric transcription whichcomprises a DNA binding domain operatively linked to a transcriptionalactivation domain. In a preferred embodiments, the fusion proteincomprises: a polypeptide selected from the group consisting of a LexADNA binding domain operatively linked to a B42 transcriptionalactivation domain operatively linked to Fus3; a Gal4 DNA binding domainoperatively linked to a B42 transcriptional activation domainoperatively linked to Fus3; and a Gal4 DNA binding domain operativelylinked to a Gal4 transcriptional activation domain II operatively linkedto Fus3.

DETAILED DESCRIPTION OF THE INVENTION

Proliferation, differentiation and death of eukaryotic cells arecontrolled by a variety of extracellular signals, such as hormones,neurotransmitters, and polypeptide factors. These diffusible ligandsallow cells to influence and be influenced by environmental cues. Thestudy of receptor-ligand interaction has revealed a great deal ofinformation about how cells respond to external stimuli, and thisknowledge has led to the development of therapeutically importantcompounds.

The present invention makes available a rapid, effective assay forscreening and identifying pharmaceutically effective compounds thatspecifically interact with and modulate the activity of a cellularreceptor, ion channel, or a surrogate of a pheromone response pathwaycomponent. The subject assay enables rapid screening of large numbers ofcompounds including, for example, small organic molecules, orpolypeptides in an expression library to identify compounds which induceor antagonize receptor bioactivity.

A particularly advantageous feature of the assay is a novel fusionprotein which comprises a protein which is responsive/sensitive toactivation of a cellular signal transduction pathway, and which isoperatively linked to a polypeptide. By virtue of its operative linkageto the protein, the polypeptide causes a detectable signal to begenerated upon stimulation/activation of the cellular signaltransduction pathway. Expression of the fusion protein of the inventionis independent of stimulation/activation of the signal transductionpathway. However, the activity of the fusion protein is signaltransduction-responsive because it comprises a signaltransduction-responsive protein the activation of which is modulateddirectly or indirectly by stimulation of pathway. Thus, the novel fusionprotein of the invention confers signal transduction responsiveness oncellular moieties (e.g., transcriptional regulatory elements, enzymes,etc.) that are not naturally responsive to activation of that signaltransduction pathway.

The assay of the present invention provides a convenient format fordiscovering drugs which can be useful to modulate cellular function, aswell as to understand the pharmacology of compounds that specificallyinteract with cellular receptors, ion channels, and components thatmodulate a surrogate of the pheromone response pathway, e.g., kinases,farnesyltransferases, and ABC transporters. Moreover, the subject assayis particularly amenable to identifying ligands, natural or artificial,for receptors and ion channels.

I. Definitions

Before further description of the invention, certain terms employed inthe specification, examples and appended claims are, for convenience,collected here.

As used herein, “recombinant cells” include any cells that have beenmodified by the introduction of heterologous DNA. Control cells includecells that are substantially identical to the recombinant cells, but donot express one or more of the proteins encoded by the heterologous DNA,e.g., do not include or express a reporter gene construct, heterologousreceptor or test polypeptide.

The term “yeast protein” as used herein refers to a protein that issensitive/responsive to, and modulated by a yeast signal transductionpathway. The yeast protein is operatively linked to a polypeptide. Theterm “yeast protein” is intended to include a full length protein, or afragment thereof, that is sensitive/responsive to the yeast signaltransduction pathway.

The terms “operatively linked”, “operably linked”, and “associated with”are used herein interchangeably and are intended to mean that moleculesare functionally coupled to each other in that the change of activity orstate of one molecule is affected by the activity or state of the othermolecule. Typically, two polypeptides are covalently attached throughpeptide bonds.

The terms “protein”, and “polypeptide” are used interchangeably herein.The term “peptide” is used herein to refer to a chain of two or moreamino acids or amino acid analogs (including non-naturally occurringamino acids), with adjacent amino acids joined by peptide (—NHCO—)bonds. Thus, the peptides of the invention include oligopeptides,polypeptides, proteins, and peptidomimetics. Methods for preparingpeptidomimetics are known in the art. In particular, a peptidomimeticcan be derived as a retro-inverso analog of the peptide. Suchretro-inverso analogs can be prepared according to methods known in theart (see, e.g., U.S. Pat. No. 4,522,752).

The term “polypeptide” as in “polypeptide operatively linked to a yeastprotein” is also intended to encompass any amino acid sequence that, byvirtue of its operative linkage to a cellular signaltransduction-responsive protein generates a change in a detectablesignal when the pathway is modulated. In certain embodiments, thepolypeptide does so by conferring signal transduction responsiveness tocellular moieties that are capable of generating a detectable signal orare capable of causing a detectable signal to be generated downstream,and that are not otherwise responsive to the signal transductionpathway. For example, in certain embodiments of the invention, the term“polypeptide” as used in this context includes, but is not limited to, atranscription factor (e.g., an endogenous yeast transcription factor), achimeric transcription factor, an enzyme (e.g., an endogenous yeastenzyme), a protein required for cell viability, or an indicator molecule(e.g., GFP).

The term “indicator molecule” as used herein refers to a polypeptidewhich provides a detectable signal, for example, green fluorescentprotein (GFP).

The term “stimulation” (as in “stimulation of a pheromoneresponse/signal transduction pathway of a yeast cell”) is intended torefer to “switching on” the yeast signal transduction cascade. Thesignal transduction cascade can be switched on by external signals thatinteract with cell receptors, e.g., ligand binding to a G-proteincoupled receptor. The term “stimulation” is also intended to encompassswitching on the yeast signal transduction cascade by any other processincluding, for example, a process similar to the process by whichphorbol esters activate the calcium dependent signal transductionpathway of T cell receptors.

The term “functionally integrated” (as in a receptor that is“functionally integrated into a signal transduction pathway in a cell”or “functionally integrated into a yeast pheromone response pathway”) isintended to refer to the ability of the receptor to be expressed at thesurface of the cell and the ability of the expressed receptor to bind tomodulators (e.g., a ligand of the receptor) and transduce signals intothe cell via components of a signal transduction pathway of the cell.For example, a G protein-coupled receptor (GPCR) which is functionallyintegrated into an endogenous pheromone response pathway of a yeast cellis expressed on the surface of the yeast cell, couples to a G protein ofthe pheromone response pathway within the yeast cell, and transduces asignal in that yeast cell upon binding of a modulator to the receptor.

The term “modulation” is intended to encompass, in its variousgrammatical forms (e.g., “modulated”, “modulation”, “modulating”, etc.),up-regulation, induction, stimulation, potentiation, localizationchanges (e.g., movement of a protein from one cellular compartment toanother) and/or relief of inhibition, as well as inhibition and/ordown-regulation.

The term “activity” as in “the activity of the yeast protein ismodulated” refers to the responsiveness or sensitivity of the yeastprotein resulting from stimulation of a yeast signal transductionpathway (e.g., the pheromone response pathway). Modulation of the yeastprotein activity may occur by any biochemical mechanism. For example,and without being bound by theory, the activity of the yeast protein maybe modulated by changing the protein from an inactive or “dormant” stateto an active or “sensitized” state. A protein may be changed from aninactive state to an active state, for example, by a conformationalchange in the protein structure. In an inactive state, the protein mayhave conformation X, but in an activated state, the protein assumesconformation Y, which enables the protein to become active/functional.Other mechanisms that involve modulating protein activity include thosein which the protein is associated with a second agent that sequestersor inhibits the protein activity. The term “agent” as used herein refersto a protein, peptide, or a factor that associates with the protein andinhibits the functional activity of the protein. Release of theassociated agent from the protein allows the protein to becomeactive/functional. Accordingly, the phrase “activity of the yeast cellis modulated” also refers to activation by release, relief, or removalof an inhibitory agent associated with the protein.

The term “directly” as in “the activity of the yeast protein ismodulated directly” refers to a process of one or more steps whereby theyeast protein is activated by stimulation of the signal transductionpathway (e.g., pheromone response pathway). For example, in oneembodiment of the invention, upon stimulation of the signal transductionpathway, the yeast protein undergoes a change in activity/stateultimately causing, for example, transcription of a gene.

The term “indirectly” as in “the activity of the yeast protein ismodulated indirectly” refers to a mechanism whereby stimulation of thesignal transduction pathway modulates the endogenous wild type yeastprotein, which in turn acts upon and modulates the activity of itscorresponding mutant yeast protein component of the fusion protein.Because the wild type yeast protein is sensitive to stimulation of thesignal transduction pathway, and the corresponding mutant yeast proteinis not sensitive, the activity of the mutant yeast protein is said to be“indirectly modulated” upon stimulation of the signal transductionpathway. The endogenous wild type yeast protein may modulate theactivity of the mutant yeast protein by any of the mechanisms describedabove with regard to direct modulation.

To summarize, the signal transduction responsive activity of the fusionprotein can be supplied in cis (from the wild type yeast proteincomponent of the chimeric fusion protein) or in trans (from theendogenous wild type yeast protein).

The term “signal transduction” is intended to encompass the processingof physical or chemical signals from the extracellular environmentthrough the cell membrane and into the cell, and may occur through oneor more of several mechanisms, such as activation/inactivation ofenzymes (such as proteases, or other enzymes which may alterphosphorylation patterns or other post-translational modifications),activation of ion channels or intracellular ion stores, effector enzymeactivation via guanine nucleotide binding protein intermediates,formation of inositol phosphate, activation or inactivation of adenylylcyclase, direct activation (or inhibition) of a transcriptional factorand/or activation. A “signal transduction pathway” refers to thecomponents involved in “signal transduction” of a particular signal intoa cell. The term “endogenous signal transduction pathway” indicates thatsome or all of the components of the signal transduction pathway arenaturally-occurring components of the cell. An example of such a pathwayis the endogenous pheromone response pathway of yeast.

The term “detecting an alteration in a signal produced by a signaltransduction pathway” (e.g., a yeast pheromone response pathway) isintended to encompass the detection of alterations in second messengersproduced upon activation of components of the signal transductionpathway, alterations in gene transcription induced upon activation ofcomponents of the signal transduction pathway, and/or alterations in theactivity of a protein(s) upon activation of components of the signaltransduction pathway. In some embodiments, the term “detecting analteration in a signal produced by an endogenous signal pathway” is not,however, intended to encompass detecting alterations in the level ofexpression of an exogenous reporter gene that has been introduced intothe cell or the activity of the reporter gene product. Moreover, theterm “detecting an alteration in a signal produced by a signaltransduction pathway” is not intended to encompass assaying general,global changes to the cell. Rather, this term indicates that a specificsignal associated with the signal transduction pathway is assayed.

As used herein, the term “extracellular signal” is intended to encompassmolecules and changes in the environment that are transducedintracellularly via cell surface proteins that interact, directly orindirectly, with the extracellular signal. An extracellular signal oreffector molecule includes any compound or substance that in some manneralters the activity of a cell surface protein. Examples of such signalsinclude, but are not limited to, molecules such as acetylcholine, growthfactors and hormones, lipids, sugars and nucleotides that bind to cellsurface and/or intracellular receptors and ion channels and modulate theactivity of such receptors and channels. The term, “extracellularsignal” also includes as yet unidentified substances that modulate theactivity of a cellular receptor, and thereby influence intracellularfunctions. Such extracellular signals are potential pharmacologicalagents that may be used to treat specific diseases by modulating theactivity of specific cell surface receptors.

The term “wild type protein” as used herein refers to unmodified,naturally occurring cellular proteins (e.g., a yeast protein) orfragments thereof.

The term “mutated protein” or “mutant protein” as used herein refers toa cellular proteins (e.g., a yeast protein), or fragment thereof, thathas been modified by addition, deletion or substitution of amino acidresidues in the protein. Preferably, the mutated protein is derived fromthe wild type protein. For example, in the case of the pheromoneresponsive wild type Fus3 yeast protein, the mutant thereof may comprisean amino acid substitution at positions 42, 180 or 182, or at positions180 and 182 in the wild type amino acid sequence.

The term “compound” as used herein (e.g., as in “test compound”) ismeant to include both exogenously added test compounds and peptidesendogenously expressed from a peptide library. For example, in certainembodiments, the reagent cell also produces the test compound which isbeing screened. The reagent cell can produce, e.g., a test polypeptide,a test nucleic acid and/or a test carbohydrate which is screened for itsability to modulate the receptor/channel activity. In such embodiments,a culture of such reagent cells will collectively provide a library ofpotential effector molecules and those members of the library whicheither agonize or antagonize the receptor or ion channel function can beselected and identified. Moreover, it will be apparent that the reagentcell can be used to detect agents which transduce a signal via thereceptor or channel of interest.

In other embodiments, the test compound is exogenously added. In suchembodiments the test compound is contacted with the reagent cell.Exemplary compounds which can be screened for activity include, but arenot limited to, peptides, nucleic acids, carbohydrates, small organicmolecules, and natural product extract libraries. In such embodiments,both compounds which agonize or antagonize the receptor- orchannel-mediated signaling function can be selected and identified.

The term “non-peptidic compound” is intended to encompass compounds thatare comprised, at least in part, of molecular structures different fromnaturally-occurring L-amino acid residues linked by natural peptidebonds. However, “non-peptidic compounds” are intended to includecompounds composed, in whole or in part, of peptidomimetic structures,such as D-amino acids, non-naturally-occurring L-amino acids, modifiedpeptide backbones and the like, as well as compounds that are composed,in whole or in part, of molecular structures unrelated tonaturally-occurring L-amino acid residues linked by natural peptidebonds, for example small organic molecules. “Non-peptidic compounds”also are intended to include natural products.

The term “receptor effector” is intended to include agonists andantagonists that modulate signal transduction via a receptor. Receptoreffector molecules are capable of binding to the receptor, though notnecessarily at the binding site of the natural ligand. Receptoreffectors can modulate signal transduction when used alone, i.e. can besurrogate ligands, or can alter signal transduction in the presence ofthe natural ligand, either to enhance or inhibit signaling by thenatural ligand. The term “antagonists” as used herein refers tomolecules that block or decrease the signal transduction activity of areceptor; e.g., they can competitively, non competitively, and/orallosterically inhibit signal transduction from the receptor.

The term “agonist” as used herein refers to agents which: induceactivation of receptor signaling pathways, e.g., such as by mimicking aligand for the receptor; potentiate the sensitivity of the receptor to aligand, e.g., lower the concentrations of ligand required to induce aparticular level of receptor-dependent signaling; or otherwise enhancethe signal transduction activity of a receptor.

The terms “receptor activator” and “surrogate ligand” as used hereinrefer to an agonist which induces signal transduction from a receptor.

“Orphan receptor” is a designation given to a receptor for which nospecific natural ligand has been described and/or for which no functionhas been determined.

The term “endogenous gene” is intended to refer to a gene in a cell thatis naturally part of the genome of the cell and which, most preferably,is present in its natural location in the genome (as opposed to“heterologous” DNA which has been introduced into the cell). Likewise,the term “endogenous protein” is intended to include proteins of a cellthat are encoded by endogenous genes of the cell.

The term “heterologous promoter” as used herein, refers to a promoterthat does not naturally regulate the gene to which the heterologouspromoter is operatively linked. For example, an endogenous yeast genethat is not normally responsive to a signal transduction pathway of theyeast cell (e.g., a yeast pheromone response pathway) can be operativelylinked to a heterologous promoter, also not normally responsive tosignals produced by the transduction pathway. A fusion protein of theinvention, which is engineered to be responsive to the signaltransduction pathway, is used to confer signal transductionresponsiveness to the endogenous yeast gene through association of thebinding site of the heterologous promoter with a region of the fusionprotein.

The term “indicator gene” as used herein refers to an expressible (e.g.,able to be transcribed and (optionally) translated) DNA sequence whichis expressed in response to activation of the fusion protein of theinvention. Exemplary indicator genes include unmodified endogenous genesoperatively linked to heterologous promoters.

The terms “reporter gene” and “reporter gene construct” are usedinterchangeably herein to refer to an indicator gene operatively linkedto at least one transcriptional regulatory sequence. Transcription ofthe reporter gene is controlled by the transcriptional regulatorysequence to which it is operatively linked. Exemplary transcriptionalcontrol sequences are promoter sequences. Examples of promoters include,but are not limited to, Gal1, Gal10, Mel and LexA operator. The activityof at least one or more of these control sequences is dependent on theactivity of a fusion protein of the current invention, in contrast tothe natural pheromone regulation of the reporter genes known in the art,(e.g., Fus1-lacZ, Fus1-HIS3, etc. see, e.g., U.S. Pat. Nos. 5,401,629and 5,691,188). A reporter gene is also meant to include apromoter-reporter gene construct which is heterologously expressed in acell.

The terms “transcriptional control element” and “transcriptionalregulatory element” are used interchangeably herein, and are intended toencompass any moiety which controls/regulates transcription of a gene towhich it is operatively linked, including, but not limited to,promoters, operators and enhancers which are responsive to the fusionproteins of the invention.

The term “chimeric nucleic acid construct” is intended to refer to anucleic acid molecule, preferably DNA, composed of at least two discretesegments. These segments are operatively linked such that uponexpression of the construct, a fusion protein is produced. The fusionprotein comprises a first polypeptide encoded by the first segment and asecond polypeptide encoded by the second segment. The first segment ofthe chimeric construct encodes a yeast protein responsive to a yeastsignal transduction pathway, for example, Fus3, or a mutation thereof,in the case of a pheromone response pathway. The second segment encodesa polypeptide that causes a detectable signal to be generated uponstimulation of the signal transduction pathway. Examples of thepolypeptides include, but are not limited to, an endogenous yeasttranscription factor, a chimeric transcription factor, an enzyme, aprotein required for yeast cell viability, or an indicator molecule.

The term “derived from” as used in the context of chimeric nucleic acidconstructs is intended to indicate that, for example, the first andsecond segments have the same or a substantially homologous nucleotidesequence as all or a part of first and second genes, respectively.Similarly, the term “derived from” as used in the context of apolypeptide sequence derived from another polypeptide is intended toindicate that the first polypeptide has the same or a substantiallyhomologous amino acid sequence as all or a part of the secondpolypeptide.

As used herein, “heterologous DNA” or “heterologous nucleic acid”includes DNA that does not occur naturally as part of the genome inwhich it is present, or which is found in a location or locations in thegenome that differs from that in which it occurs in nature. HeterologousDNA is DNA that is not naturally occurring in that position or is notendogenous to the cell into which it is introduced, but has beenobtained from another cell. Generally, although not necessarily, suchDNA encodes proteins that are not normally produced by the cell in whichit is expressed. Heterologous DNA can be from the same species, althoughin preferred embodiments, it is from a different species. Inparticularly preferred embodiments, it is mammalian, e.g., human.Heterologous DNA may also be referred to as foreign DNA. Any DNA thatone of skill in the art would recognize or consider as heterologous orforeign to the cell in which it is expressed is herein encompassed bythe term heterologous DNA. Examples of heterologous DNA include, but arenot limited to, DNA that encodes test polypeptides, receptors, reportergenes, transcriptional and translational regulatory sequences, orselectable or traceable marker proteins, such as a protein that confersdrug resistance.

The terms “heterologous protein”, “recombinant protein”, and “exogenousprotein” are used interchangeably throughout the specification and referto a polypeptide which is produced by recombinant DNA techniques,wherein generally, DNA encoding the polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. That is, the polypeptide isexpressed from a heterologous nucleic acid.

The term “substantially homologous”, when used in connection with aminoacid sequences, refers to sequences which are substantially identical toor similar in sequence, giving rise to a homology in conformation andthus to similar biological activity. The term is not intended to imply acommon evolution of the sequences.

Typically, “substantially homologous” sequences are at least 50%, morepreferably at least 80%, identical in sequence, at least over anyregions known to be involved in the desired activity. Most preferably,no more than five residues, other than at the termini, are different.Preferably, the divergence in sequence, at least in the aforementionedregions, is in the form of “conservative modifications”.

To determine the percent homology of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).For example, the length of a reference sequence aligned for comparisonpurposes is at least 30%, preferably at least 40%, more preferably atleast 50%, even more preferably at least 60%, and even more preferablyat least 70%, 80%, or 90% of the length of the reference sequence (e.g.,when aligning a second sequence to the first amino acid sequence whichhas for example 100 amino acid residues, at least 30, preferably atleast 40, more preferably at least 50, even more preferably at least 60,and even more preferably at least 70, 80 or 90 amino acid residues arealigned). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch (J. Mol.Biol. (48):444–453 (1970)) algorithm which has been incorporated intothe GAP program in the GCG software package, using either a Blossom 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4, and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment,the percent identity between two nucleotide sequences is determinedusing the GAP program in the GCG software package, using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80, and a length weight of1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity betweentwo amino acid or nucleotide sequences is determined using the algorithmof E. Meyers and W. Miller (CABIOS, 4:11–17 (1989)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to identify, for example, other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403–10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to NIP2b, NIP2cL, and NIP2cS nucleic acid molecules of theinvention: BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to NIP2b, NIP2cL, and NIP2cS protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389–3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

As used herein, “cell surface receptor” refers to molecules that occuron the surface of cells, interact with the extracellular environment,and transmit or transduce the information regarding the environmentintracellularly in a manner that may modulate intracellular secondmessenger activities or transcription of specific promoters, resultingin transcription of specific genes. A “heterologous receptor” is aspecific embodiment of a “heterologous protein”, wherein theheterologous receptor is encoded by heterologous DNA and, uponexpression of this heterologous DNA in a recombinant cell, theheterologous receptor is expressed in the recombinant cell.

The term “pheromone system protein surrogate” (abbreviated as “PSPsurrogate”) is intended to refer to a heterologous protein in a yeastcell which is functionally homologous to a yeast protein of thepheromone response pathway (i e., the PSP surrogate is functionallyintegrated into the yeast pheromone system pathway). Examples of PSPsurrogates, and methods of preparing yeast cells comprising such PSPsurrogates, are described in detail in PCT Publication WO 94/23025.Preferred PSP surrogates include G protein-coupled receptors, Gproteins, proteases, kinases, farnesyltransferases,carboxymethyltransferases, ABC transporters and cyclins.

The term “autocrine cell”, as used herein, refers to a cell whichproduces a substance which can stimulate a receptor located on or withinthe same cell as that which produces the substance. For example,wild-type yeast MATα and MATa cells are not autocrine. However, a yeastcell which produces both α-factor and α-factor receptor, or botha-factor and a-factor receptor, in functional form, is autocrine. Byextension, cells which produce a peptide which is being screened for theability to activate a receptor (e.g., by activating a G protein-coupledreceptor) and also express the receptor are called “autocrine cells”. Insome instances, such cells can also be referred to as “putativeautocrine cells” since some of the cells will express peptides from thelibrary which will not activate the receptor which is expressed. In alibrary of such cells, in which a multitude of different peptides areproduced, it is likely that one or more of the cells will be “autocrine”in the stricter sense of the term.

II. General Overview of Assay

As set out above, the invention relates to methods for identifyingcompounds from among a set or collection or library of one or morecompounds that modulate the activity of a signal transduction pathway ina cell. The pathway may be an endogenous signal transduction pathwaywithin the cell (for example, the pheromone response pathway in a yeastcell), or may comprise one or more surrogate components which functionin place of a natural component of the pathway.

The method of the present invention makes use of a cell that comprises apolypeptide made responsive to the signal transduction pathway throughoperative linkage to a component of the pathway, such that thepolypeptide is capable of causing a detectable signal to be generatedupon activation of the signal transduction pathway. In accordance withthe method, the cell is contacted with a test compound, and themodulatory effect of the compound on the activity of the signaltransduction pathway is assessed.

Test compounds which act as agonists are detected as compounds whichcause an increase in detectable signal as compared with the signal inthe absence of the test compound. In another aspect, the effect of thetest compounds on cells that are essentially identical except for thepresence or absence of a target protein (e.g., a receptor, an ionchannel, or a signal transduction pathway component surrogate) can bedetected. Compounds which act as antagonists are detected as those whichcause a decrease in the detectable signal generated by an agonist or anatural stimulator of signal transduction pathway when compared with thesame cell in the absence of the test compound.

Alternatively, the target specificity of the test compound may beassessed by comparing the detectable signals generated in cells whichdiffer only in the surrogate component of the signal transductionpathway. For example, cells which comprise different functionallycoupled G protein-coupled receptors (GPCRs) may be compared in this way.Differences in detectable signal may then be ascribed to the GPCRs andmay be distinguished from effects due to components present in eachcell. In another embodiment, the cells may differ in that one cellcomprises a functional surrogate signal transduction component (e.g.,mammalian GPCR) whereas the other is identical except that the naturalcomponent is substituted for the functional surrogate.

In certain embodiments, a test compound is exogenously added, and itsability to modulate the activity of the target receptor or ion channelis scored in the assay. In other embodiments, the cells are engineeredto express additionally a test polypeptide which can be assayed for itsability to interact with the receptor or ion channel. In thoseembodiments, the assay provides a population of cells which express alibrary of peptides which include potential receptor/channel effectors,and those peptides of the library which either agonize or antagonize thereceptor or channel function can be selected and identified by sequence.

The ability of particular compounds to modulate the signal transductionactivity of target receptor or channel can be scored for by detecting upor down-regulation of a detection signal. For example, second messengergeneration (e.g. GTPase activity, phospholipid hydrolysis, or proteinphosphorylation patterns) can be measured directly. In otherembodiments, transcription of an endogenous gene or activity of anendogenous protein is used as a detectable readout.

Alternatively, the use of an indicator gene can provide a convenientreadout. In other embodiments, a detection means consists of a reportergene. In any event, a statistically significant change in the detectionsignal can be used to facilitate identification of compounds whichmodulate receptor or ion channel activities.

By this method, compounds which induce a signal pathway from aparticular receptor or channel can be identified. If a test compounddoes not appear to induce the activity of the receptor/channel protein,the assay may be repeated and modified by the introduction of a step inwhich the reagent cell is first contacted with a known activator of thetarget receptor/channel to induce signal transduction, and the testcompound can be assayed for its ability to inhibit the activatedreceptor/channel, e.g., to identify antagonists. In yet otherembodiments, batteries of compounds can be screened for agents whichpotentiate the response to a known activator of the receptor.

The method of the present invention is useful for identifying compoundsthat interact with any receptor protein whose activity ultimatelyinduces a signal transduction cascade in the host cell which can beexploited to produce a detectable signal. In particular, the assays canbe used to test functional ligand-receptor or ligand-ion channelinteractions for cell surface-localized receptors and channels, and alsofor cytoplasmic and nuclear receptors. As described in more detailbelow, the subject assay can be used to identify effectors of, forexample, G protein-coupled receptors, receptor tyrosine kinases,cytokine receptors, and ion channels, as well as steroid hormone, orother nuclear receptors. In certain embodiments the method describedherein is used for identifying ligands for “orphan receptors” for whichno ligand is known.

In embodiments utilizing an “autocrine cell” of the present invention,and in which cell surface receptors are the assay targets, it will bedesirable for each of the peptides of the peptide library to include asignal sequence for secretion. In certain embodiments the expression ofsuch a signal sequence may ensure appropriate transport of the peptideto the endoplasmic reticulum, the golgi, and ultimately to the cellsurface. When a yeast cell is the host cell, in certain embodiments, thesignal sequence will transport peptides to the periplasmic space,however, such transport may not be necessary to achieve autocrinestimulation.

Any transfectable cell that can express the desired cell surface proteinin a manner such the protein functions to transduce intracellularly anextracellular signal may be used. Similarly, any cell surface proteinthat is known to those of skill in the art or that may be identified bythose of skill in the art may used in the assay. The cell surfaceprotein may be endogenously expressed on the selected cell or it may beexpressed from cloned DNA.

III. Chimeric Nucleic Acid Constructs Expressing Fusion Proteins thatConfer Signal Transduction Responsiveness

In one aspect, the invention provides chimeric nucleic acid constructsthat express fusion proteins that are responsive to cellular signaltransduction pathways. Expression of the fusion protein need not beconstitutive, although it is important to note that the activity of thefusion protein, and not its expression, is responsive to the signaltransduction pathway. The constructs comprise a first segment comprisinga nucleotide sequence encoding a cellular protein that is responsive toa cellular signal transduction pathway, wherein the activity of theprotein is modulated directly or indirectly upon stimulation of acellular signal transduction pathway, and a second segment comprising anucleotide sequence encoding a polypeptide that causes a detectablesignal to be generated upon modulation of the transduction pathwayresponsive protein. The fusion protein expressed by the chimericconstruct comprises the signal transduction-responsive proteinoperatively linked to the polypeptide. Thus, the fusion protein isresponsive to activation/stimulation of the signal transduction pathwayby virtue of the component comprising the cellular protein that isresponsive to the cellular signal transduction pathway. The polypeptidecan be a cellular transcription factor, a chimeric transcription factor,an enzyme, a protein required for cell viability, or an indicatormolecule.

In a preferred embodiment, the cell is a yeast cell. Yeast proteins thatare responsive to yeast signal transduction pathways include, but arenot limited to, Fus3, Hog1, Kss1, Mpk1, Smk1, Bem1, Cdc24, Cdc42, Dig1,Dig2, Far1, Gpa1, Msg5, Ste4, Ste5, Ste7, Ste11, Ste12, Ste18, Ste20 andSst2. In another preferred embodiment, the yeast signal transductionpathway is the yeast pheromone response pathway.

Yeast proteins that are sensitive/responsive to the pheromone responsepathway include kinases that are homologous to the highly conservedfamily of kinases called mitogen activated protein (MAP) kinases.Members of the MAP kinase family are activated by a variety ofextracellular agents and influence cellular proliferation anddifferentiation. In Saccharomyces cerevisiae five MAP kinase genehomologs have been identified, (Davis et. al., (1995) Mol. Reprod. Dev.42, 459–467).

Preferred pheromone sensitive/responsive yeast proteins include Fus3,Kss1, Bem1, Cdc24, Cdc42, Dig1, Dig2, Far1, Gpa1, Msg5, Ste4, Ste5,Ste7, Ste11, Ste12, Ste18, Ste20 and Sst2. Especially preferred for thepractice of the invention is Fus3, which is activated during thepheromone response pathway and, based on the high degree of homologywith MAP kinase, may function in a similar kinase cascade (see, e.g.,Errede et. al., (1995) Mol. Reprod. and Dev. 42, 477–45 and Gartner et.al., (1992) Genes and Dev. 6, 1280–1292). The gene encoding Fus3 wascloned by Fujimura (who referred to the gene as Dac2) (see Fujimura(1990) Curr. Genet. 18, 395–400). Standard molecular biology proceduresare used for making fusion proteins comprising Fus3 operatively linkedto a polypeptide, as described in Example 1. Far1, Ste5, Ste11 andSte12, other pheromone-responsive yeast proteins, are also particularlypreferred.

In one embodiment, the fusion protein comprises a wild type yeastprotein that is responsive to a yeast signal transduction pathway. Inanother embodiment, the fusion protein comprises a mutant yeast proteinderived from the wild type protein. For example, in the case of Fus3, apheromone responsive yeast protein, the fusion protein can comprise amutant Fus3 protein comprising amino acid substitutions at positions 42,180, 182, or at positions 180 and 182 as compared to the wild type Fus3amino acid sequence. Exemplary amino acid substitutions in the Fus3 wildtype amino acid sequence, include, but are not limited to, Lys42Arg,Thr180Val, Thr180Glu, Tyr182Val, and Tyr182Asp. The production of mutantpheromone responsive Fus3 yeast proteins is described in Example 2.

In one embodiment, the second segment of the chimeric nucleic acidconstruct comprises a nucleotide sequence that encodes a cellulartranscription factor. The fusion protein expressed by the chimericnucleic acid construct comprises the signal transduction responsiveprotein operatively linked to the transcription factor. Polypeptideswhich can function as transcription factors to activate transcription inprokaryotic cells are well known in the art. Any wild type transcriptionfactor of interest may be operatively linked to the signal transductionresponsive protein for use in the method of the invention. In the caseof a yeast signal transduction pathway, transcription factors suitablefor use in the present invention include, but are not limited to, theendogenous yeast transcription factors Ste12, Gal4, Pho4, Gcn4, Hap1,Adr1, Ace2, Cup2, Swi5 and Bas1.

In another embodiment, the second segment of the chimeric nucleic acidconstruct comprises a nucleotide sequence that encodes a chimerictranscription factor. The second segment comprises a third segmentcomprising a nucleotide sequence encoding a DNA binding domain, and afourth segment comprising a nucleotide sequence encoding atranscriptional activation domain. Thus, the fusion protein expressed bythe chimeric nucleic acid construct comprises a signaltransduction-responsive protein operatively linked to a chimerictranscription factor comprising a DNA binding domain operatively linkedto a transcriptional activation domain.

Chimeric transcription factors can be constructed using standardmolecular biology techniques as described in Example 1. The DNA bindingdomain and the transcriptional activation domain of the chimerictranscription factor can be derived either from the same protein, e.g.,a prokaryotic protein, or from different proteins, e.g., a prokaryoticprotein and a eukaryotic protein, or two different proteins from thesame organism.

DNA binding domains that bind to specific regulatory sequences are alsowell known in the art (see, e.g., Keegan et al., (1988), Science, 231,699–704; Hope et al., (1986) Cell, 46, 885–894 and Ma et al., (1987)Cell 51, 113–119). A selected transcriptional activation domain can bepaired with a DNA binding domain to activate transcription (Brent etal., (1985) Cell 43, 729–736; see also U.S. Pat. No. 4,833,080 to Brentet al. which discloses regulation of eukaryotic gene expression using atwo component chimeric fusion protein consisting of a DNA binding domainand a transcriptional activation domain) providing the nucleotidesequence to be transcribed is operatively linked to a promoter sequencerecognized by the selected DNA binding domain. Residues 1–147 of Gal4confer sequence specific DNA binding (See Carey et al., (1989) J. Mol.Biol. 209, 423–432). Residues 1–87 of the bacteria repressor LexA alsoconfer sequence specific DNA binding activity (See Olesen et al., (1990)Genes Dev. 4, 1714–1729 and Brent et al., (1985) Cell 43, 729–236). DNAbinding domains that can be used in the invention include, but are notlimited to, LexA, Gal4, Adr1, Ace2, Cup2, Bas1, Gcn4, Swi5, Pho4, Hap1,and LacI.

Transcriptional activation domains of many DNA binding proteins havebeen described and have been shown to retain their activation functionwhen the domain is transferred to a heterologous polypeptide.Transcriptional activation domains found within various proteins havebeen grouped into categories based upon similar structural features.Types of transcriptional activation domains include acidic transcriptionactivation domains, proline-rich transcription activation domains,serine/threonine-rich transcription activation domains andglutamine-rich transcription activation domains. Examples of acidictranscriptional activation domains include VP16 and amino acid residues753–881 of GAL4. Examples of proline-rich activation domains includeamino acid residues 399–499 of CTF/NF1 and amino acid residues 31–76 ofAP2. Examples of serine/threonine-rich transcription activation domainsinclude amino acid residues 1–427 of ITF1 and amino acid residues 2–451of ITF2. Examples of glutamine-rich activation domains include aminoacid residues 175–269 of Oct1 and amino acid residues 132–243 of Sp1.The amino acid sequences of each of the above described regions, and ofother useful transcriptional activation domains, are disclosed inSeipel, K. et al. (EMBO J. (1992) 13:4961–4968).

In addition to previously described transcriptional activation domains,novel transcriptional activation domains, which can be identified bystandard techniques, are within the scope of the invention. Thetranscriptional activation ability of a polypeptide can be assayed byoperatively linking a transcriptional activation domain to a DNA bindingdomain to form a chimeric transcription factor. The activity of thetranscription factor can be determined using the assays described inExample 1, and the amount of transcription of a target sequence can bedetermined. Preferred transcriptional activation domains include but arenot limited to the B42, Gal4, Adr1 Ace2, Cup2, Bas1, Gcn4, Swi5, Pho4,Hap1, VP16, and Ste 12. The B42, VP16, Gal4, and Ste12 transcriptionalactivation domains are particularly preferred.

In the case of activation of a yeast pheromone response pathway in ayeast cell, the activity of the pheromone responsive yeast protein ismodulated directly or indirectly, thereby causing the chimerictranscription factor to activate transcription of a gene that produces adetectable protein. For example, in a preferred embodiment, the firstsegment of the construct encodes wild type Fus3 and the second segmentencodes a chimeric transcription factor; that is, the second segmentcomprises a third segment that encodes the entire prokaryotic proteinLexA as the DNA binding domain, and a fourth segment that encodes an 88amino acid segment of an acidic E. coli peptide (B42AD) as thetranscriptional activation domain. Upon expression of the chimericconstruct, signaling through the pheromone response pathway modulatesFus3 thereby activating the LexA-B42AD component (the “chimerictranscription factor”) of the fusion protein, to which component Fus3 isoperatively linked. The chimeric transcription factor activatestranscription of the gene which is operatively linked to a promoterwhich contains the binding sites for LexA and LexA operators. Thus, inthe case of the lacZ indicator gene under the control of the LexAoperator (p8op-lacZ), the LexA-B42AD-Fus3 fusion protein binds to LexAbinding site in the promoter and induces expression of the lacZ gene.β-Galactosidase is the detectable signal resulting from expression ofthe lacZ gene. Exemplary chimeric transcription factors include, but arenot limited to, LexA-B42AD, Gal4DBD-B42AD, Gal4DBD-VP16AD andGal4DBD-Gal4ADII.

In certain embodiments of the invention wherein the second segment ofthe chimeric nucleic acid construct encodes a cellular transcriptionfactor, or a chimeric transcription factor, the fusion protein resultingfrom expression of the construct can be used to confer signaltransduction-responsiveness on indicator genes, including heterologousgenes as well as endogenous yeast genes, that are not normallyresponsive to the signal transduction pathway, as described in Examples1 and 2. Examples of indicator genes suitable for use in accordance withthe invention include ADE1, ADE2, ADE3, ADE4, ADE5, ADE7, ADE8, ASP3,ARG1, ARG3, ARG4, ARG5, ARG6, ARG8, ARO2, ARO7, BAR1, CAT, CHO1, CYS3,GAL1, GAL7, GAL10, GFP, HIS1, HIS3, HIS4, HIS5, HOM3, HOM6, ILV1, ILV2,ILV5, INO1, INO2, INO4, lacZ, LEU1, LEU2, LEU4, luciferase, LYS2, MAL,MEL MET2, MET3, MET4, MET8, MET9, MET14, MET16, MET19, OLE1, PHO5, PRO1,PRO3, THR1, THR4, TRP1, TRP2, TRP3, TRP4, TRP5, URA1, URA2, URA3, URA4,URA5 and URA10. Preferred indicator genes include CAT, GAL1, GAL7,GAL10, GFP, HIS3, lacZ, luciferase, LEU2, MEL, PHO5, and URA3.

In practicing one embodiment of the invention, a reporter gene constructis inserted into the reagent cell that will produce a detection signalupon activation of the chimeric fusion protein. Typically, the reportergene construct will include an indicator gene in operative linkage withone or more transcriptional control elements, the activity of which isindirectly regulated by the signal transduction activity of the targetreceptor, with the level of expression of the reporter gene providingthe receptor-dependent detection signal. The amount of transcriptionfrom the indicator gene may be measured using any method known to thoseof skill in the art to be suitable.

Transcriptional control elements for use in the reporter geneconstructs, or for modifying the genomic locus of an indicator geneinclude, but are not limited to, promoters, enhancers, and operators,the activities of which are responsive to the polypeptide comprising thetranscription factor/chimeric transcription factor component of thechimeric fusion protein of the invention. That is, the transcriptionalcontrol elements are not normally responsive to the signal transductionpathway of the cell, but are made responsive by interaction with thefusion protein.

In another embodiment, the second segment of the nucleic acid constructof the invention encodes a polypeptide that comprises an enzyme. Thefusion protein expressed by the chimeric construct comprises the yeastprotein operatively linked to the enzyme. Upon stimulation of the signaltransduction pathway, the yeast protein is modulated and confers anability to the enzyme to directly produce a detectable signal. Byoperatively linking the yeast cell protein to the enzyme, the method ofthe invention eliminates the requirement for a reporter gene construct.Instead, stimulation of the signal transduction pathway is monitoreddirectly as a result of the enzyme producing an end product whichprovides the detectable signal. For example, the enzyme can be aconventional enzyme that catalyzes the conversion of a substrate to aproduct and thereby generate a detectable signal, such as, e.g., achange in color, fluorescence, or luminescence. Examples of such enzymesinclude CAT (chloramphenicol acetyl transferase) (Alton and Vapnek(1979), Nature 282: 864–869); luciferase, β-galactosidase; fireflyluciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725–737); bacterialluciferase (Engebrecht and Silverman (1984), P.N.A.S. 1: 4154–4158;Baldwin et al. (1984), Biochemistry 23: 3663–3667); alkaline phosphatase(Toh et al. (1989) Eur. J. Biochem. 182: 231–238, Hall et al. (1983) J.Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase(Cullen and Malim (1992) Methods in Enzymol. 216:362–368); β-lactamase,etc.

Alternatively, the enzyme can be a metabolic enzyme that relieves acell's nutritional requirement and/or provides resistance to a drug. Forexample, in one embodiment, the imidazoleglycerol phosphate dehydratase(IGP dehydratase) (ie., the His3 enzyme) can be used in the chimericfusion protein of the invention. In the simplest case, the cell isauxotrophic for histidine (requires histidine for growth) in the absenceof activation. Activation of the His3 enzyme, through association withthe signal transduction responsive protein component of the fusionprotein, causes the cell to become prototrophic for histidine (does notrequire histidine). Thus the selection is for growth in the absence ofhistidine. Since only a few molecules per cell of IGP dehydratase arerequired for histidine prototrophy, the assay is very sensitive.

In yet another embodiment, the second segment of the nucleic acidconstruct of the invention encodes a polypeptide that comprises aprotein required for cell viability. The fusion protein expressed by thechimeric construct comprises the signal transduction responsive proteinoperatively linked to the protein required for cell viability. Detectionof cell growth and viability can be used as an effective screeningprocedure. In a yeast embodiment, to determine cell viability, apheromone responsive yeast protein is operatively linked to apolypeptide that confers cell growth or viability to the yeast cell (SeeExample 3). When the pheromone response pathway is stimulated, the yeastprotein confers a signal pathway dependent prototrophy on the yeast cellto enable the yeast cell to grow on media plates on which it would nottypically be able to grow. As exemplified in Example 3, a yeast protein(e.g., Ste11) that is operatively linked to His3. This chimericconstruct can be transformed into a suitable yeast strain, e.g., astrain auxotrophic for histidine. Upon stimulation of the pheromoneresponse pathway, the chimeric construct confers pheromone pathwaydependent histidine prototrophy on the yeast cell which allows thestrain to grow on histidine minus media plates.

Examples of polypeptides required for cell growth include, but are notlimited to, Ade1, Ade2, Ade3, Ade4, Ade5, Ade7, Ade8, Asp3, Arg1, Arg3,Arg4, Arg5, Arg6, Arg8, Aro2, Aro7, Bar1, Cho1, Cys3, Gal1, Gal7, Gal10,His1, His3, His4, His5, Hom3, Hom6, Ilv1, Ilv2, Ilv5, Ino1, Ino2, Ino4,Leu1, Leu2, Leu4, Lys2, Mal, Mel, Met2, Met3, Met4, Met8, Met9, Met14,Met16, Met19, Ole1, Pho5, Pro1, Pro3, Thr1, Thr4, Trp1, Trp2, Trp3,Trp4, Trp5, Ura1, Ura2, Ura3, Ura4, Ura5, Ura10, Cdc25, Cyr1 and Ras.Particularly preferred polypeptides that confer growth and cellviability include Gal1, His3, Leu2, Mel, Ura3, Cdc25, Cyr1 and Ras.

Another example of a protein that is required for cell viability is Ras.In mammalian cells, Ras is a membrane associated GTPase that functionsas a molecular switch to activate intracellular mitogen-activatedprotein kinase (MAPK) cascades and other effector pathways in responseto a cellular signal. Activation of Ras into its GTP-bound conformationis directly controlled by specific guanine-nucleotide exchange factors(GEF's), which catalyze GDP release (See e.g., Fan et al. (1988) Curr.Biol. 13: 935–938). Several Ras-specific GEFs that relate to the yeastprotein Cdc25, have been described. Proteins involved in the Ras cellsignaling pathway or cascade are art recognized. See, e.g., Murray, A.and Hunt, T. eds. The Cell Cycle: An Introduction (W.H. Freeman andCompany, New York) pp. 109–110. Briefly, the Ras cell signaling cascadebegins with cell activation, e.g., cell activation by a growth factor,and activation of the growth factor receptor. Receptor binding leads tothe binding of adaptor proteins, for example, GRB2. The adaptor proteinsactivate guanine nucleotide-exchange proteins and GTPase activatingproteins, e.g., p120-GAP, which, in turn, activate small G proteins suchas Ras. Ras, in turn, induces activation and phosphorylation of Raf,MEK, p44 and p42 MAP kinases (ERK1 and ERK2). Raf is the first member ofthe protein kinase cascade which ultimately leads to the phosphorylationand activation of MAP kinase. Activation of MAP kinase leads to itstranslocation into the nucleus where it induces transcription.

The activities of RAS genes are essential for viability of a yeast cell.Although a yeast cell is still viable upon deletion of either the RAS1or RAS2 genes, deletion of both genes is lethal. Moreover, deletion ofthe RAS2 gene is lethal for a cell grown in media consisting ofnonfermentable carbon sources, such as glycerol and ethanol. Inaddition, expression of the mammalian Ras protein is capable of rescuingthe yeast cell from lethality . (T. Kataoka, et al., (1984) Cell 37:437;T. Kataoka, et al., (1985) Cell 40:19; D. DeFeo-Jones et al., (1985)Science 228:179; and K. Tatchell et al. (1985) PNAS 82:3785). Effectiveinteraction between Ras and its downstream target, adenylyl cyclase,which is localized on the membrane, is essential. Thus, the membraneassociation of Ras proteins is crucial to cell viability (T. Toda et al.(1985) Cell 40:27; J E Buss et al. (1989) Science, 243:1600; and J FHancock et al., (1989) Cell, 57:1167).

Operatively linking a yeast protein to a Ras protein whose localizationto the membrane is dependent on the activation of a signal transductionpathway thus confers cell viability to a cell in which one or more RASgenes has been inactivated. Thus, in one embodiment, the yeast protein,e.g., a pheromone responsive yeast protein, is operatively linked toRas. Upon stimulation of the pheromone response pathway, the activity ofthe yeast protein is modulated, which in turn modulates the activity ofRas. Ras subsequently activates other members of the cascade andultimately confers cell growth. In a preferred embodiment, the pheromoneresponsive protein is Ste5.

The foregoing embodiments demonstrate that overall, the inventionprovides a rapid, reproducible, robust assay system that makes use of afusion protein, the activity of which is responsive to a cellular signaltransduction pathway (e.g., the pheromone response pathway in yeastcells), to confer signal transduction responsiveness on cellularmoieities, including, but not limited to, indicator genes, transcriptionregulatory elements, enzymes, and proteins required for cell viabilitythat are not otherwise responsive to the signal transduction pathway.

IV. Host Cells

Suitable host cells for generating the subject assay includeprokaryotes, yeast, or higher eukaryotic cells, including plant andanimal cells, especially mammalian cells. Prokaryotes include gramnegative or gram positive organisms. Examples of suitable mammalian hostcell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman (1981) Cell 23:175) CV-1 cells (ATCC CCL 70), L cells, C127,3T3, Chinese hamster ovary (CHO), HeLa, HEK-293, SWISS 3T3, and BHK celllines.

If yeast cells are used, the yeast may be of any species which arecultivable and in which an exogenous receptor can be made to engage theappropriate signal transduction machinery of the host cell. Suitablespecies include Kluyveromyces lactis, Schizosaccharomyces pombe, andUstilago maydis; Saccharomyces cerevisiae is preferred. Other yeastwhich can be used in practicing the present invention are Neurosporacrassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris,Candida tropicalis, and Hansenula polymorpha. The term “yeast”, as usedherein, includes not only yeast in a strictly taxonomic sense, i.e.,unicellular organisms, but also yeast-like multicellular fungi orfilamentous fungi.

It will be understood that to achieve selection or screening, the hostcell must have an appropriate phenotype. For example, generating apheromone-responsive chimeric HIS3 gene in a yeast that has a wild-typeHIS3 gene would frustrate genetic selection. Thus, to achievenutritional selection, an auxotrophic strain is preferred.

A variety of complementations for use in the subject assay can beconstructed. Indeed, many yeast genetic complementations with mammaliansignal transduction proteins have been described in the art. Forexample, Mosteller et al. (1994) Mol. Cell Biol. 14:1104–12 demonstratesthat human Ras proteins can complement loss of ras mutations in S.cerevisiae. Moreover, Toda et al. (1986) Princess Takamatsu Symp 17:253–60 have shown that human Ras proteins can complement the loss ofRas1 and Ras2 proteins in yeast, and hence are functionally homologous.Both human and yeast Ras proteins can stimulate the magnesium andguanine nucleotide-dependent adenylate cyclase activity present in yeastmembranes. Ballester et al. (1989) Cell 59: 681–6 describe a vector toexpress the mammalian GAP protein in the yeast S. cerevisiae. Whenexpressed in yeast, GAP inhibits the function of the human Ras protein,and complements the loss of IRA1. IRA1 is a yeast gene that encodes aprotein with homology to GAP and acts upstream of Ras. Mammalian GAP cantherefore function in yeast and interact with Ras yeast. Wei et al.(1994) Gene 151: 279–84 describes that a human Ras-specific guaninenucleotide-exchange factor, Cdc25GEF, can complement the loss of Cdc25function in S. cerevisiae. Martegani et al. (1992) EMBO J 11: 2151–7describe the cloning by functional complementation of a mouse cDNAencoding a homolog of Cdc25, a Saccharomyces cerevisiae Ras activator.Vojtek et al. (1993) J. Cell Sci. 105: 777–85 and Matviw et al. (1992)Mol. Cell Biol. 12: 5033–40 describe how a mouse CAP protein, e.g., anadenylyl cyclase associated protein associated with Ras-mediated signaltransduction, can complement defects in S. cerevisiae. Papasavvas et al.(1992) Biochem. Biophys. Res. Commun. 184:1378–85 also suggest thatinactivated yeast adenyl cyclase can be complemented by a mammalianadenyl cyclase gene. Hughes et al. (1993) Nature 364: 349–52 describethe complementation of byr1 in fission yeast by mammalian MAP kinase(MEK). Parissenti et al. (1993) Mol Cell Endocrinol 98: 9–16 describethe reconstitution of bovine protein kinase C (PKC) in yeast. The Ca²⁺and phospholipid-dependent Ser/Thr kinase PKC plays important roles inthe transduction of cellular signals in mammalian cells. Marcus et al.(1995) P.N.A.S. 92: 6180–4 suggest the complementation of shkl nullmutations in S. pombe by either the structurally related S. cerevisiaeSte20 or mammalian p65PAK protein kinases.

“Inactivation”, with respect to genes of the host cell, means thatproduction of a functional gene product is prevented or inhibited.Inactivation may be achieved by deletion of the gene, mutation of thepromoter so that expression does not occur, or mutation of the codingsequence so that the gene product is inactive. Inactivation may bepartial or total.

“Complementation”, with respect to genes of the host cell, means that atleast partial function of an inactivated gene of the host cell issupplied by an exogenous nucleic acid. For instance, yeast cells can be“mammalianized”, and even “humanized”, by complementation of receptorand signal transduction proteins with mammalian homologs. To illustrate,inactivation of a yeast Byr2/Ste11 gene can be complemented byexpression of a human MEKK gene.

In certain embodiments (particularly those in which an autocrine peptidelibrary is employed), the growth arrest consequent to activation of thepheromone response pathway is an undesirable effect since cells thatbind agonists stop growing while surrounding cells that fail to bindpeptides will continue to grow. The cells of interest, then, will beovergrown or their detection obscured by the background cells,confounding identification of the compound of interest. To overcome thisproblem the present invention teaches engineering the cell such that: 1)growth arrest does not occur as a result of exogenous signal pathwayactivation (e.g., by inactivation of the FAR1 gene); and/or 2) aselective growth advantage is conferred by activating the pathway (See,e.g., Example 3).

It is desirable that the exogenous receptor be exposed on a continuingbasis to the test compound. Unfortunately, this is likely to result indesensitization of the pheromone response pathway to the stimulus. Forexample, the mating signal transduction pathway is known to becomedesensitized by several mechanisms including pheromone degradation andmodification of the function of the receptor, G proteins and/ordownstream elements of the pheromone signal transduction by the productsof the SST2, STE50, AFR1 (Konopka, J. B. (1993) Mol. Cell. Biol.13:6876–6888) and SGV1, MSG5, and SIG1 genes. Selected mutations inthese genes can lead to hypersensitivity to pheromone and an inabilityto adapt to the presence of pheromone. For example, introduction ofmutations that interfere with function into strains expressingheterologous G protein-coupled receptors constitutes a significantimprovement on wild type strains and enables the development ofextremely sensitive bioassays for compounds that interact with thereceptors. Other mutations, e.g., STE50, SGV1, BAR1, STE2, STE3, PIK1,MSG5, SIG1 and AFT1, have the similar effect of increasing thesensitivity of the bioassay. Thus desensitization may be avoided bymutating (which may include deleting) the SST2 gene so that it no longerproduces a functional protein, or by mutating one of the other geneslisted above.

V. Expression Systems

Ligating a polynucleotide coding sequence into a gene construct, such asan expression vector, and transforming or transfecting into hosts,either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic(bacterial cells), are standard procedures used in producing otherwell-known proteins, including sequences encoding exogenous receptor andpeptide libraries. Similar procedures, or modifications thereof, can beemployed to prepare recombinant reagent cells of the present inventionby tissue-culture technology in accord with the subject invention.

In general, it will be desirable that the vector be capable ofreplication in the host cell. It may be DNA which is integrated into thehost genome, and thereafter is replicated as a part of the chromosomalDNA, or it may be DNA which replicates autonomously, as in the case of aplasmid. In the latter case, the vector will include an origin ofreplication which is functional in the host. In the case of anintegrating vector, the vector may include sequences which facilitateintegration, e.g., sequences homologous to host sequences, or encodingintegrases.

Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are known in the art, andare described in, for example, Powels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, New York, 1985). Mammalian expressionvectors may comprise non-transcribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, other 5′ or 3′ flanking nontranscribed sequences, 5′ or 3′nontranslated sequences, such as necessary ribosome binding sites, apoly-adenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

Preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

Transcriptional and translational control sequences in expressionvectors to be used in transforming mammalian cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al. (1978) Nature 273:111). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the BglI site locatedin the viral origin of replication is included. Exemplary vectors can beconstructed as disclosed by Okayama and Berg (1983, Mol. Cell Biol.3:280). A useful system for stable high level expression of mammalianreceptor cDNAs in C127 murine mammary epithelial cells can beconstructed substantially as described by Cosman et al. (1986, Mol.Immunol. 23:935). Other expression vectors for use in mammalian hostcells are derived from retroviruses.

In other embodiments, the use of viral transfection can provide stablyintegrated copies of the expression construct. In particular, the use ofretroviral, adenoviral or adeno-associated viral vectors is contemplatedas a means for providing a stably transfected cell line which expressesan exogenous receptor, and/or a polypeptide library.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs in S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. Moreover, if yeast are used as a host cell, it will be understoodthat the expression of a gene in a yeast cell requires a promoter whichis functional in yeast. Suitable promoters include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Req. 7, 149 (1968); and Holland et al. Biochemistry 17, 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedmetallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well asenzymes responsible for maltose, galactose and melibiose utilization.Finally, promoters that are active in only one of the two haploid matingtypes may be appropriate in certain circumstances. Among thesehaploid-specific promoters, the pheromone promoters MFa1 and MFα1 are ofparticular interest.

In some instances, it may be desirable to use insect cells as the hostcells. In such embodiments, recombinant polypeptides can be expressed bythe use of a baculovirus expression system. Examples of such baculovirusexpression systems include pVL-derived vectors (such as pVL1392, pVL1393and pVL941), pAcUW-derived vectors (such as pAcUW1), andpBlueBac-derived vectors (such as the β-gal containing pBlueBac III).

In constructing suitable expression plasmids, the termination sequencesassociated with these genes, or with other genes which are efficientlyexpressed in yeast, may also be ligated into the expression vector 3′ ofthe heterologous coding sequences to provide polyadenylation andtermination of the mRNA.

In certain embodiments, the host cell harbors a reporter gene constructcontaining an indicator gene in operative linkage with one or moretranscriptional regulatory elements that associate with the chimericfusion protein of the invention. Exemplary indicator genes includeenzymes, such as luciferase, phosphatase, or β-galactosidase which canproduce a spectrometrically active label, e.g., changes in color,fluorescence or luminescence, or a gene product which alters a cellularphenotype, e.g., cell growth, drug resistance or auxotrophy. Forexample, in certain embodiments, the indicator gene encodes a geneproduct selected from the group consisting of chloramphenicol acetyltransferase, β-galactosidase and secreted alkaline phosphatase. In stillother embodiments, the indicator gene encodes a gene product whichconfers a growth signal. In yet another embodiment, the indicator geneencodes a gene product that permits prototrophic growth, or that conferssensitivity to drugs for counterselection purposes, e.g., canavanine orcycloheximide.

VI. Receptors

Receptor proteins (e.g., pheromone system protein surrogates) for use inthe present invention can be any receptor or ion channel which interactswith an extracellular molecule (i.e., hormone, growth factor, peptide,ion) to modulate a signal in the cell. To illustrate, the receptor canbe a cell surface receptor or, in other embodiments, an intracellularreceptor. In certain embodiments, the receptor is a cell surfacereceptor, such as: a receptor tyrosine kinase, e.g., an EPH receptor; anion channel; a cytokine receptor; a chemokine receptor; a growth factorreceptor; or a G-protein coupled receptor, such as a chemoattractantpeptide receptor, a neuropeptide receptor, a light receptor, aneurotransmitter receptor, or a polypeptide hormone receptor. In apreferred embodiment, the pheromone system protein surrogate to beassayed is selected from the group consisting of G protein-coupledreceptors, G proteins, proteases, kinases, farnesyltransferases,carboxymethyltransferases, ABC transporters and cyclins. The differentgroups of receptors are described in detail below.

In addition, the subject assay can be used to identify ligands for anorphan receptor, i.e., a receptor with no known ligand, regardless ofthe class of receptors to which it belongs.

In those embodiments wherein the target receptor is a cell surfacereceptor and the cell expresses a peptide library, it may be desirable,in certain embodiments, for the peptides in the library to express asignal sequence to ensure that they are processed in the appropriatesecretory pathway and thus are available to interact with receptors onthe cell surface.

A. Cytokine Receptors

In one embodiment, the target receptor is a cytokine receptor. Cytokinesare a family of soluble mediators of cell-to-cell communication thatincludes interleukins, interferons, and colony-stimulating factors. Thecharacteristic features of cytokines lie in their functional redundancyand pleiotropy. Most of the cytokine receptors that constitute distinctsuperfamilies do not possess intrinsic protein tyrosine kinase domains,yet receptor stimulation usually invokes rapid tyrosine phosphorylationof intracellular proteins, including the receptors themselves. Manymembers of the cytokine receptor superfamily activate the Jak proteintyrosine kinase family, with resultant phosphorylation of the STATtranscriptional activator factors. IL-2, IL-4, IL-7 and Interferon γhave all been shown to activate Jak kinases (Frank et al. (1995) Proc.Natl. Acad. Sci. USA 92:7779–7783); Scharfe et al. (1995) Blood86:2077–2085); (Bacon et al. (1995) Proc. Natl. Acad. Sci. USA92:7307–7311); and (Sakatsume et al. (1995) J. Biol. Chem.270:17528–17534). Events downstream of Jak phosphorylation have alsobeen elucidated. For example, exposure of T lymphocytes to IL-2 has beenshown to lead to the phosphorylation of signal transducers andactivators of transcription (STAT) proteins STAT1α, STAT2β, and STAT3,as well as of two STAT-related proteins, p94 and p95. The STAT proteinswere found to translocate to the nucleus and to bind to a specific DNAsequence, thus suggesting a mechanism by which IL-2 may activatespecific genes involved in immune cell function (Frank et al. supra).Jak3 is associated with the gamma chain of the IL-2, IL-4, and IL-7cytokine receptors (Fujii et al. (1995) Proc. Natl. Acad. Sci.92:5482–5486) and (Musso et al. (1995) J. Exp. Med. 181:1425–1431). TheJak kinases have also been shown to be activated by numerous ligandsthat signal via cytokine receptors such as, growth hormone anderythropoietin and IL-6 (Kishimoto (1994) Stem cells Suppl. 12:37–44).

B. Nuclear Receptors.

In another embodiment, the target receptor is a nuclear receptor. Thenuclear receptors may be viewed as ligand-dependent transcriptionfactors. These receptors provide a direct link between extracellularsignals, mainly hormones, and transcriptional responses. Theirtranscriptional activation function is regulated by endogenous smallmolecules, such as steroid hormones, vitamin D, ecdysone, retinoic acidsand thyroid hormones, which pass readily through the plasma membrane andbind their receptors inside the cell (Laudet and Adelmant (1995) CurrentBiology 5:124). The majority of these receptors appear to contain threedomains: a variable amino terminal domain; a highly conserved,DNA-binding domain and a moderately conserved, carboxyl-terminalligand-binding domain (Power et al. (1993) Curr. Opin. Cell Biol.5:499–504). Examples include the estrogen, progesterone, androgen,thyroid hormone and mineralocorticoid receptors. In addition to theknown steroid receptors, at least 40 orphan members of this superfamilyhave been identified. (Laudet et al., (1992) E.M.B.O. J. 11:1003–1013).There are at least four groups of orphan nuclear receptors representedby NGF1, FTZ-F1, Rev-erbs, and RARs, which are by evolutionarystandards, only distantly related to each other (Laudet et al. supra).While the steroid hormone receptors bind exclusively as homodimers to apalindrome of their hormone responsive element other nuclear receptorsbind as heterodimers. Interestingly, some orphan receptors bind asmonomers to similar response elements and require for their function aspecific motif that is rich in basic amino-acid residues and is locatedcorboxy-terminal to the DNA-binding domain (Laudet and Adelmant supra.)

C. Receptor Tyrosine Kinases.

In another embodiment, the target receptor is a receptor tyrosinekinase. The receptor tyrosine kinases can be divided into five subgroupson the basis of structural similarities in their extracellular domainsand the organization of the tyrosine kinase catalytic region in theircytoplasmic domains. Sub-groups I (epidermal growth factor (EGF)receptor-like), II (insulin receptor-like) and the eph/eck familycontain cysteine-rich sequences (Hirai et al., (1987) Science238:1717–1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol.10:6316–6324). The functional domains of the kinase region of thesethree classes of receptor tyrosine kinases are encoded as a contiguoussequence (Hanks et al. (1988) Science 241:42–52). Subgroups III(platelet-derived growth factor (PDGF) receptor-like) and IV (thefibro-blast growth factor (FGF) receptors) are characterized as havingimmunoglobulin (Ig)-like folds in their extracellular domains, as wellas having their kinase domains divided in two parts by a variablestretch of unrelated amino acids (Yanden and Ullrich (1988) supra andHanks et al. (1988) supra).

The family with by far the largest number of known members is the EPHfamily. Since the description of the prototype, the EPH receptor (Hiraiet al. (1987) Science 238:1717–1720), sequences have been reported forat least ten members of this family, not counting apparently orthologousreceptors found in more than one species. Additional partial sequences,and the rate at which new members are still being reported, suggest thefamily is even larger (Maisonpierre et al. (1993) Oncogene 8:3277–3288;Andres et al. (1994) Oncogene 9:1461–1467; Henkemeyer et al. (1994)Oncogene 9:1001–1014; Ruiz et al. (1994) Mech. Dev. 46:87–100; Xu et al.(1994) Development 120:287–299; Zhou et al. (1994) J. Neurosci. Res.37:129–143; and references in Tuzi and Gullick (1994) Br. J. Cancer69:417–421). Remarkably, despite the large number of members in the EPHfamily, all of these molecules were identified as orphan receptorswithout known ligands.

As used herein, the terms “EPH receptor” or “EPH-type receptor” refer toa class of receptor tyrosine kinases, comprising at least elevenparalogous genes, though many more orthologs exist within this class,e.g. homologs from different species. EPH receptors, in general, are adiscrete group of receptors related by homology and easily recognizable,e.g., they are typically characterized by an extracellular domaincontaining a characteristic spacing of cysteine residues near theN-terminus and two fibronectin type III repeats (Hirai et al. (1987)Science 238:1717–1720; Lindberg et al. (1990) Mol. Cell Biol.10:6316–6324; Chan et al. (1991) Oncogene 6:1057–1061; Maisonpierre etal. (1993) Oncogene 8:3277–3288; Andres et al. (1994) Oncogene9:1461–1467; Henkemeyer et al. (1994) Oncogene 9:1001–1014; Ruiz et al.(1994) Mech. Dev. 46:87–100; Xu et al. (1994) Development 120:287–299;Zhou et al. (1994) J. Neurosci. Res. 37:129–143; and references in Tuziand Gullick (1994) Br. J. Cancer 69:417–421). Exemplary EPH receptorsinclude the eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyro1, tyro4,tyro5, tyro6, tyro11, cek4, cek5, cek6, cek7, cek8, cek9, cek10, bsk,rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2, pagliaccio, htk, erk and nukreceptors. The term “EPH receptor” refers to the membrane form of thereceptor protein, as well as soluble extracellular fragments whichretain the ability to bind the ligand of the present invention.

In exemplary embodiments, the detection signal is provided by detectingphosphorylation of intracellular proteins, e.g., MEKKs, MEKs, or Mapkinases, or by the use of reporter constructs or indicator genes whichinclude transcriptional regulatory elements responsive to c-fos and/orc-jun.

D. G Protein-Coupled Receptors.

One family of signal transduction cascades found in eukaryotic cellsutilizes heterotrimeric “G proteins.” Many different G proteins areknown to interact with receptors. G protein signaling systems includethree components: the receptor itself, a GTP-binding protein (Gprotein), and an intracellular target protein. The cell membrane acts asa switchboard. Messages arriving through different receptors can producea single effect if the receptors act on the same type of G protein. Onthe other hand, signals activating a single receptor can produce morethan one effect if the receptor acts on different kinds of G proteins,or if the G proteins can act on different effectors.

In their resting state, the G proteins, which consist of alpha (α), beta(β) and gamma (γ) subunits, are complexed with the nucleotide guanosinediphosphate (GDP) and are in contact with receptors. When a hormone orother first messenger binds to a receptor, the receptor changesconformation and this alters its interaction with the G protein. Thisspurs the α subunit to release GDP, and the more abundant nucleotideguanosine triphosphate (GTP), replaces it, activating the G protein. TheG protein then dissociates to separate the a subunit from the stillcomplexed beta and gamma subunits. Either the Gα subunit, or the Gβγcomplex, depending on the pathway, interacts with an effector. Theeffector (which is often an enzyme) in turn converts an inactiveprecursor molecule into an active “second messenger,” which may diffusethrough the cytoplasm, triggering a metabolic cascade. After a fewseconds, the Gα converts the GTP to GDP, thereby inactivating itself.The inactivated Gα may then reassociate with the Gβγ complex.

Hundreds, if not thousands, of receptors convey messages throughheterotrimeric G proteins, of which at least 17 distinct forms have beenisolated. Although the greatest variability has been seen in the αsubunit, several different β and γ structures have been reported. Thereare, additionally, many different G protein-dependent effectors.

Most G protein-coupled receptors are comprised of a single protein chainthat is threaded through the plasma membrane seven times. Such receptorsare often referred to as seven-transmembrane receptors (STRs). More thana hundred different STRs have been found, including many distinctreceptors that bind the same ligand, and there are likely many more STRsawaiting discovery.

In addition, STRs have been identified for which the natural ligands areunknown; these receptors are termed “orphan” G protein-coupledreceptors, as described above. Examples include receptors cloned byNeote et al. (1993) Cell 72, 415; Kouba et al. FEBS Lett. (1993) 321,173; and Birkenbach et al. (993) J. Virol. 67, 2209.

The “exogenous receptors” of the present invention may be any Gprotein-coupled receptor, preferably exogenous to the cell, which is tobe genetically engineered for the purpose of the present invention. Thisreceptor may be a plant or animal cell receptor. Screening for bindingto plant cell receptors may be useful in the development of, e.g.,herbicides. In the case of an animal receptor, it may be of invertebrateor vertebrate origin. If an invertebrate receptor, an insect receptor ispreferred, and would facilitate development of insecticides. Thereceptor may also be a vertebrate, more preferably a mammalian, stillmore preferably a human, receptor. The exogenous receptor is alsopreferably a seven transmembrane segment receptor.

Known ligands for G protein coupled receptors include: purines andnucleotides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin and thelike; biogenic amines (and related natural ligands), such as5-hydroxytryptamine, acetylcholine, dopamine, adrenaline, histamine,noradrenaline, tyramine/octopamine and other related compounds; peptidessuch as adrenocorticotrophic hormone (acth), melanocyte stimulatinghormone (msh), melanocortins, neurotensin (nt), bombesin and relatedpeptides, endothelins, cholecystokinin, gastrin, neurokinin b (nk3),invertebrate tachykinin-like peptides, substance k (nk2), substance p(nk1), neuropeptide y (npy), thyrotropin releasing-factor (trf),bradykinin, angiotensin ii, beta-endorphin, c5a anaphalatoxin,calcitonin, chemokines (also called intercrines), corticotrophicreleasing factor (crf), dynorphin, endorphin, fmlp and other formylatedpeptides, follitropin (fsh), fungal mating pheromones, galanin, gastricinhibitory polypeptide receptor (gip), glucagon-like peptides (glps),glucagon, gonadotropin releasing hormone (gnrh), growth hormonereleasing hormone(ghrh), insect diuretic hormone, interleukin-8,leutropin (1h/hcg), met-enkephalin, opioid peptides, oxytocin,parathyroid hormone (pth) and pthrp, pituitary adenylyl cyclaseactivating peptide (pacap), secretin, somatostatin, thrombin,thyrotropin (tsh), vasoactive intestinal peptide (vip), vasopressin,vasotocin; eicosanoids such as ip-prostacyclin, pg-prostaglandins,tx-thromboxanes; retinal based compounds such as vertebrate 11-cisretinal, invertebrate 11-cis retinal and other related compounds; lipidsand lipid-based compounds such as cannabinoids, anandamide,lysophosphatidic acid, platelet activating factor, leukotrienes and thelike; excitatory amino acids and ions such as calcium ions andglutamate.

Preferred G protein coupled receptors include, but are not limited to:α1A-adrenergic receptor, α1B-adrenergic receptor, α2-adrenergicreceptor, α2B-adrenergic receptor, β1-adrenergic receptor, β2-adrenergicreceptor, β3-adrenergic receptor, m1 acetylcholine receptor (AChR), m2AChR, m3 AChR, m4 AChR, m5 AChR, D1 dopamine receptor, D2 dopaminereceptor, D3 dopamine receptor, D4 dopamine receptor, D5 dopaminereceptor, A1 adenosine receptor, A2a adenosine receptor, A2b adenosinereceptor, A3 adenosine receptor, 5-HT1a receptor, 5-HT1b receptor,5HT1-like receptor, 5-HT1d receptor, 5HT1d-like receptor, 5HT1d betareceptor, substance K (neurokinin A) receptor, fMLP receptor (FPR),fMLP-like receptor (FPRL-1), angiotensin II type 1 receptor, endothelinETA receptor, endothelin ETB receptor, thrombin receptor, growthhormone-releasing hormone (GHRH) receptor, vasoactive intestinal peptidereceptor, oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3,cannabinoid receptor, follicle stimulating hormone (FSH) receptor,leutropin (LH/HCG) receptor, thyroid stimulating hormone (TSH) receptor,thromboxane A2 receptor, platelet-activating factor (PAF) receptor, C5aanaphylatoxin receptor, CXCR1 (IL-8 receptor A), CXCR2 (IL-8 receptorB), Delta Opioid receptor, Kappa Opioid receptor, mip-1alpha/RANTESreceptor (CRR1), Rhodopsin, Red opsin, Green opsin, Blue opsin,metabotropic glutamate mGluR1-6, histamine H2 receptor, ATP receptor,neuropeptide Y receptor, amyloid protein precursor receptor,insulin-like growth factor II receptor, bradykinin receptor,gonadotropin-releasing hormone receptor, cholecystokinin receptor,melanocyte stimulating hormone receptor, antidiuretic hormone receptor,glucagon receptor, and adrenocorticotropic hormone II receptor. Inaddition, there are at least five receptors (CC and CXC receptors)involved in HIV viral attachment to cells. The two major co-receptorsfor HIV are CXCR4, (fusin receptor, LESTR, SDF1 receptor) and CCR5(m-trophic). More preferred receptors include the following humanreceptors: melatonin receptor 1a, galanin receptor 1, neurotensinreceptor, adenosine receptor 2a, somatostatin receptor 2 andcorticotropin releasing factor receptor 1. Melatonin receptor 1a isparticularly preferred. Other G protein coupled receptors (GPCRs) areknown in the art. The term “receptor,” as used herein, encompasses bothnaturally occurring and mutant receptors.

Many of these G protein-coupled receptors, like the yeast a- andα-factor receptors, contain seven hydrophobic amino acid-rich regionswhich are assumed to lie within the plasma membrane. Specific human Gprotein-coupled STRs for which genes have been isolated and for whichexpression vectors could be constructed include those listed herein andothers known in the art. Thus, the gene would be operably linked to apromoter functional in the cell to be engineered and to a signalsequence that also functions in the cell. For example in the case ofyeast, suitable promoters include STE2, STE3, Gal1, and Gal10. Suitablesignal sequences include those of STE2, STE3 and of other genes whichencode proteins secreted by yeast cells. Preferably, when a yeast cellis used, the codons of the gene would be optimized for expression inyeast. See Hoekema et al., (1987) Mol. Cell. Biol., 7:2914–24; Sharp, etal., (1986) 14:5125–43.

The homology of STRs is discussed in Dohlman et al., Ann. Rev. Biochem.,(1991) 60:653–88. When STRs are compared, a distinct spatial pattern ofhomology is discernible. The transmembrane domains are often the mostsimilar, whereas the N- and C-terminal regions, and the cytoplasmic loopconnecting transmembrane segments V and VI are more divergent.

The functional significance of different STR regions has been studied byintroducing point mutations (both substitutions and deletions) and byconstructing chimeras of different but related STRs. Synthetic peptidescorresponding to individual segments have also been tested for activity.Affinity labeling has been used to identify ligand binding sites.

In certain embodiments, if the wild-type exogenous G protein-coupledreceptor cannot be made functional in yeast, it may be mutated for thispurpose. A comparison would be made of the amino acid sequences of theexogenous receptor and of the yeast receptors, and regions of high andlow homology identified. Trial mutations would then be made todistinguish regions involved in ligand or G protein binding, from thosenecessary for functional integration in the membrane. The exogenousreceptor would then be mutated in the latter region to more closelyresemble the yeast receptor, until functional integration was achieved.If this were insufficient to achieve functionality, mutations would nextbe made in the regions involved in G protein binding. Mutations would bemade in regions involved in ligand binding only as a last resort, andthen an effort would be made to preserve ligand binding by makingconservative substitutions whenever possible. For example, the V–VI loopof a heterologous G protein coupled receptor could be replaced with thatof the yeast STE2 or STE3 receptor).

In yet another embodiment, a compatible G protein can be provided. Acompatible G protein for use in the instant assays can include aheterologous or chimeric G protein subunit (or subunits) such as thosedescribed in the art (see e.g., PCT PCT/US94/03143). Preferably, theyeast genome is modified so that it is unable to produce the yeastreceptors which are homologous to the exogenous receptors in functionalform.

F. Chemoattractant Receptors

An exemplary GPCR is the N-formyl peptide receptor (FPR), a classicexample of a calcium mobilizing GPCR expressed by neutrophils and otherphagocytic cells of the mammalian immune system (Snyderman et al. (1988)In Inflammation: Basic Principles and Clinical Correlates, pp. 309–323).N-formyl peptides of bacterial origin bind to the receptor and engage acomplex activation program that results in directed cell movement,release of inflammatory granule contents, and activation of a latentNADPH oxidase which is important for the production of metabolites ofmolecular oxygen. This pathway initiated by receptor-ligand interactionis critical in host protection from pyogenic infections. Similar signaltransduction occurs in response to the inflammatory peptides C5a andInterleukin 8.

Two other formyl peptide receptor like (FPRL) genes have been clonedbased on their ability to hybridize to a fragment of the FPR cDNA codingsequence. These have been named FPRL1 (Murphy et al. (1992) J. BiolChem. 267:7637–7643) and FPRL2 (Ye et al. (1992) Biochem Biophys Res.Comm. 184:582–589). FPRL2 was found to mediate calcium mobilization inmouse fibroblasts transfected with the gene and exposed to formylpeptide. In contrast, although FPRL1 was found to be 69% identical inamino acid sequence to FPR, it did not bind prototype N-formyl peptidesligands when expressed in heterologous cell types. This lead to thehypothesis of the existence of an as yet unidentified ligand for theFPRL1 orphan receptor (Murphy et al. supra).

VII. Test Compounds

A. Exogenously Added Compounds

A recent trend in medicinal chemistry includes the production ofmixtures of compounds, referred to as libraries. While the use oflibraries of peptides is well established in the art, new techniqueshave been developed which have allowed the production of mixtures ofother compounds, such as benzodiazepines (Bunin et al. 1992. J. Am.Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA:6909), peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678)oligocarbamates (Cho et al. 1993. Science 261:1303), and hydantoins(DeWitt et al. supra). Rebek et al. have described an approach for thesynthesis of molecular libraries of small organic molecules with adiversity of 104–105 (Carell et al. 1994. Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).

The compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. Anticancer Drug Des. 1997.12:145).

In one embodiment, the test compound is a peptide or peptidomimetic. Inanother, preferred embodiment, the compounds are small, organicnon-peptidic compounds.

Other exemplary methods for the synthesis of molecular libraries can befound in the art, for example in: Erb et al. 1994. Proc. Natl. Acad.Sci. USA 91:11422; Horwell et al. 1996 Immunopharmacology 33:68; and inGallop et al. 1994. J. Med. Chem. 37:1233. In addition, libraries suchas those described in the commonly owned applications U.S. Ser. No.08/864,241 now abandonded, U.S. Ser. No. 08/864,240 now U.S. Pat. No.6,037,340 and U.S. Ser. No. 08/835,623 now abandonded can be used toprovide compounds for testing in the present invention.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412–421), or on beads (Lam (1991) Nature354:82–84), chips (Fodor (1993) Nature 364:555–556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865–1869) or on phage(Scott and Smith (1990) Science 249:386–390; Devlin (1990) Science249:404–406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378–6382;Felici (1991) J. Mol. Biol. 222:301–310; and Ladner, supra).

Other types of peptide libraries may also be expressed, see, e.g., U.S.Pat. Nos. 5,270,181 and 5,292,646; and PCT publication WO94/02502). Instill another embodiment, the combinatorial polypeptides are producedfrom a cDNA library.

Exemplary compounds which can be screened for activity include, but arenot limited to, peptides, nucleic acids, carbohydrates, small organicmolecules, and natural product extract libraries. In such embodiments,both compounds which agonize or antagonize the receptor- orchannel-mediated signaling function can be selected and identified.

If a test compound fails to stimulate the activity of a receptor, theassay may be repeated and modified by the introduction of a step inwhich the reagent cell is first contacted with a known activator of thetarget receptor/channel to induce signal transduction, and the testcompound can be assayed for its ability to inhibit the activatedreceptor/channel, e.g., to identify antagonists. In yet otherembodiments, batteries of compounds can be screened for agents whichpotentiate the response to a known activator of the receptor.

B. Peptide Libraries

In certain embodiments, yeast cells can be engineered to produce thecompounds to be tested. This assay system has the advantage ofincreasing the effective concentration of the compound to be tested. Inone embodiment, a method such as that described in WO 94/23025 can beutilized.

Other methods can also be used. As mentioned above, peptide librariesare systems which simultaneously display, in a form which permitsinteraction with a target, a highly diverse and numerous collection ofpeptides. Many of the systems known in the art for presentation ofpeptides in a library are limited in terms of the maximum length of thepeptide or the composition of the peptide (e.g., Cys excluded). Stericfactors, such as the proximity of a support, may interfere with binding.Usually, the screening is for binding in vitro to an artificiallypresented target, not for activation or inhibition of a cellular signaltransduction pathway in a living cell. Although a cell surface receptormay be used as a target, the screening will not reveal whether thebinding of the peptide caused an allosteric change in the conformationof the receptor.

The Ladner et al. patent, U.S. Pat. No. 5,096,815, describes a method ofidentifying novel proteins or polypeptides with a desired DNA bindingactivity. Semi-random “variegated”) DNA encoding a large number ofdifferent potential binding proteins is introduced, in expressible form,into suitable yeast cells. The target DNA sequence is incorporated intoa genetically engineered operon such that the binding of the protein orpolypeptide will prevent expression of a gene product that isdeleterious to the gene under selective conditions. Cells which survivethe selective conditions are thus cells which express a protein whichbinds the target DNA. While it is taught that yeast cells may be usedfor testing, bacterial cells are preferred. The interactions between theprotein and the target DNA occur only in the cell (and then only in thenucleus), not in the periplasm or cytoplasm, and the target is a nucleicacid, and not a receptor protein. Substitution of random peptidesequences for functional domains in cellular proteins permits somedetermination of the specific sequence requirements for theaccomplishment of function. Though the details of the recognitionphenomena which operate in the localization of proteins within cellsremain largely unknown, the constraints on sequence variation ofmitochondrial targeting sequences and protein secretion signal sequenceshave been elucidated using random peptides (Lemire et al., J. Biol.Chem. (1989) 264, 20206 and Kaiser et al. (1987) Science 235:312,respectively).

In certain embodiments of the instant invention, the compounds testedare in the form of peptides from a peptide library. The peptide libraryof the present invention takes the form of a cell culture, in whichessentially each cell expresses one, and usually only one, peptide ofthe library. While the diversity of the library is maximized if eachcell produces a peptide of a different sequence, it is usually prudentto construct the library so there is some redundancy. Depending on size,the combinatorial peptides of the library can be expressed as is, or canbe incorporated into larger fusion proteins. The fusion protein canprovide, for example, stability against degradation or denaturation, aswell as a secretion signal if secreted. In an exemplary embodiment of alibrary for intracellular expression, e.g., for use in conjunction withintracellular target receptors, the polypeptide library is expressed asthioredoxin fusion proteins (see, for example, U.S. Pat. Nos. 5,270,181and 5,292,646; and PCT publication WO94/02502). The combinatorialpeptide can be attached to one of the termini of the thioredoxinprotein, or, for short peptide libraries, inserted into the so-calledactive loop.

In one embodiment, the peptide library is derived to express acombinatorial library of polypeptides which are not based on any knownsequence, nor derived from cDNA. That is, the sequences of the libraryare largely random. In preferred embodiments, the combinatorialpolypeptides are in the range of 3–100 amino acids in length, morepreferably at least 5–50, and even more preferably at least 10, 13, 15,20 or 25 amino acid residues in length. Preferably, the polypeptides ofthe library are of uniform length. It will be understood that the lengthof the combinatorial peptide does not reflect any extraneous sequenceswhich may be present in order to facilitate expression, e.g., such assignal sequences or invariant portions of a fusion protein.

In another embodiment, the peptide library is derived to express acombinatorial library of polypeptides which are based at least in parton a known polypeptide sequence or a portion thereof (not a cDNAlibrary). That is, the sequences of the library are semi-random, beingderived by combinatorial mutagenesis of a known sequence. (See, e.g,Ladner et al. PCT publication WO 90/02909; Garrard et al., PCTpublication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007–16010; Griffths et al. (1993) E.M.B.O. J 12:725–734; Clacksonet al. (1991) Nature 352:624–628; and Barbas et al. (1992) P.N.A.S.89:4457–4461). Accordingly, polypeptides which are known ligands for atarget receptor can be mutagenized by standard techniques to derive avariegated library of polypeptide sequences which can further bescreened for agonists and/or antagonists. This library can be expressedin a reagent cell of the present invention, and other receptoractivators can be isolated from the library. This has permitted theidentification of even more potent FPRL-1 surrogate ligands (Klein etal., supra).

Alternatively, the library can be expressed under conditions wherein thecells are in contact with the original tridecapeptide, e.g., the FPRL-1receptor is being induced by that surrogate ligand. Peptides from anexpressed library can be isolated based on their ability to potentiatethe induction, or to inhibit the induction, caused by the surrogateligand. The latter of course will identify potential antagonists ofchemoattractant receptors. In still other embodiments, the surrogateligand can be used to screen exogenous compound libraries (peptide andnon-peptide) which, by modulating the activity of the identifiedsurrogate, will presumably also similarly effect the native ligand'seffect on the target receptor. In such embodiments, the surrogate ligandcan be applied to the cells, though is preferably produced by thereagent cell, thereby providing an autocrine cell.

In still another embodiment, the combinatorial polypeptides are producedfrom a cDNA library.

In a preferred embodiment of the present invention, the yeast cellscollectively produce a “peptide library”, preferably including at least10³ to 10⁷ different peptides, so that diverse peptides may besimultaneously assayed for the ability to interact with the exogenousreceptor. In an especially preferred embodiment, at least some peptidesof the peptide library are secreted into the periplasm, where they mayinteract with the “extracellular” binding site(s) of an exogenousreceptor. They thus mimic more closely the clinical interaction of drugswith cellular receptors. This embodiment optionally may be furtherimproved (in assays not requiring pheromone secretion) by preventingpheromone secretion, and thereby avoiding competition between thepeptide and the pheromone for signal peptidase and other components ofthe secretion system.

In certain embodiments of the present invention, the peptides of thelibrary are encoded by a mixture of DNA molecules of different sequence.Each peptide-encoding DNA molecule is ligated with a vector DNA moleculeand the resulting recombinant DNA molecule is introduced into a yeastcell. Since it is a matter of chance which peptide encoding DNA moleculeis introduced into a particular cell, it is not predictable whichpeptide that cell will produce. However, based on a knowledge of themanner in which the mixture was prepared, one may make certainstatistical predictions about the mixture of peptides in the peptidelibrary.

The peptides of the library can be composed of constant and variableresidues. If the nth residue is the same for all peptides of thelibrary, it is said to be constant. If the nth residue varies, dependingon the peptide in question, the residue is a variable one. The peptidesof the library will have at least one, and usually more than one,variable residue. A variable residue may vary among any of two to alltwenty of the genetically encoded amino acids; the variable residues ofthe peptide may vary in the same or different manner. Moreover, thefrequency of occurrence of the allowed amino acids at a particularresidue position may be the same or different. The peptide may also haveone or more constant residues.

There are two principal ways in which to prepare the required DNAmixture. In one method, the DNAs are synthesized a base at a time. Whenvariation is desired, at a base position dictated by the Genetic Code, asuitable mixture of nucleotides is reacted with the nascent DNA, ratherthan the pure nucleotide reagent of conventional polynucleotidesynthesis.

The second method provides more exact control over the amino acidvariation. First, trinucleotide reagents are prepared, eachtrinucleotide being a codon of one (and only one) of the amino acids tobe featured in the peptide library. When a particular variable residueis to be synthesized, a mixture is made of the appropriatetrinucleotides and reacted with the nascent DNA. Once the necessary“degenerate” DNA is complete, it must be joined with the DNA sequencesnecessary to assure the expression of the peptide, as discussed in moredetail below, and the complete DNA construct must be introduced into theyeast cell.

C Periplasmic Secretion

In those embodiments of the invention in which yeast cells are used asthe host cell and the compounds tested are endogenously expressed from alibrary, it will be noted that the yeast cell is bounded by a lipidbilayer called the plasma membrane. Between this plasma membrane and thecell wall is the periplasmic space. Peptides secreted by yeast cellscross the plasma membrane through a variety of mechanisms and therebyenter the periplasmic space. The secreted peptides are then free tointeract with other molecules that are present in the periplasm ordisplayed on the outer surface of the plasma membrane. The peptides theneither undergo re-uptake into the cell, diffuse through the cell wallinto the medium, or become degraded within the periplasmic space.

The test polypeptide library may be secreted into the periplasm by anyof a number of exemplary mechanisms, depending on the nature of theexpression system to which they are linked. In one embodiment, thepeptide may be structurally linked to a yeast signal sequence, such asthat present in the α-factor precursor, which directs secretion throughthe endoplasmic reticulum and Golgi apparatus. Because this is the sameroute that the receptor protein follows in its journey to the plasmamembrane, opportunity exists in cells expressing both the receptor andthe peptide library for a specific peptide to interact with the receptorduring transit through the secretory pathway. This has been postulatedto occur in mammalian cells exhibiting autocrine activation. Suchinteraction could yield activation of the response pathway duringtransit, which would still allow identification of those cellsexpressing a peptide agonist. For situations in which peptideantagonists to externally applied receptor agonist are sought, thissystem would still be effective, since both the peptide antagonist andreceptor would be delivered to the outside of the cell in concert. Thus,those cells producing an antagonist would be selectable, since thepeptide antagonist would be properly and timely situated to prevent thereceptor from being stimulated by the externally applied agonist.

An alternative mechanism for delivering peptides to the periplasmicspace is to use the ATP-dependent transporters of the STE6/MDR1 class.This transport pathway and the signals that direct a protein or peptideto this pathway are not as well characterized as is the endoplasmicreticulum-based secretory pathway. Nonetheless, these transportersapparently can efficiently export certain peptides directly across theplasma membrane, without the peptides having to transit the ER/Golgipathway. It is anticipated that at least a subset of peptides can besecreted through this pathway by expressing the library in context ofthe α-factor prosequence and terminal tetrapeptide. The possibleadvantage of this system is that the receptor and peptide do not comeinto contact until both are delivered to the external surface of thecell. Thus, this system strictly mimics the situation of an agonist orantagonist that is normally delivered from outside the cell. Use ofeither of the described pathways is within the scope of the invention.

The present invention does not require periplasmic secretion, or, ifsuch secretion is provided, any particular secretion signal or transportpathway.

EXEMPLIFICATION

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand are not intended to limit the invention.

Example 1 Fusion Proteins Comprising a Pheromone Sensitive/ResponsiveYeast Protein

This example demonstrates the use of recombinant yeast cellsincorporating fusion proteins which comprise a chimeric transcriptionfactor operatively linked to a pheromone sensitive/responsive yeastprotein to provide a rapid detection assay for stimulation of thepheromone response pathway by an external signal (e.g., a ligand). Thechimeric transcription factor comprises a DNA binding domain and atranscriptional activation domain. Principally, any activation domaincan be paired with any DNA binding domain to activate transcription. Inthe present example, the entire prokaryotic protein LexA was used as theDNA binding domain, and an 88 amino acid segment of an acidic E. colipeptide (B42AD) was used as the transcriptional activation domain forthe chimeric transcription factor. Both LexA and B42AD were obtainedfrom Clontech Laboratories, Inc. The indicator gene was lacZ which wasunder the control of 8 copies of LexA operator (p8op-lacZ, ClontechLaboratories, Inc.). This chimeric transcription factor constitutivelyactivates transcription in yeast (data not shown). Expression of the LBFchimeric transcription factor complements fus3 deletion in yeast cells(data not shown). Fus3 was used as the pheromone sensitive yeast proteinand was fused downstream of the chimeric transcription factor togenerate a LexA-B42AD-Fus3 fusion protein, referred to as “LBF”. Thegene encoding Fus3 was cloned and sequenced by Fujimura (See Fujimura(1990) Curr. Genet. 18, 395–400). Ligand-activation of G-protein coupledreceptors in yeast cells was measured using this LBF chimerictranscription factor plus p8op-lacZ reporter gene construct, asdescribed below.

Construction of Plasmids:

DNA encoding an 88 amino acid fragment of an acidic E. coli peptide thatactivates transcription in yeast (B42AD) was cloned in pLexA (ClonetechLaboratories, Inc) along with the HA epitope tag for PCR amplificationusing: Template: pB42AD from Clontech Laboratories, Inc

(SEQ ID NO: 1) Primer1: CTAGGGATCCGGGAGAGGCATAATCTGGCAC (SEQ ID NO: 2)Primer2: GATCGAATTCGGTATCAATAAAGATATCGAGGAGTGC.

The resulting PCR product was purified, digested with EcoRI and BamHIand ligated in pLexA (Clontech Laboratories, Inc) that was digested withEcoRI and BamHI enzymes to generate Cp5650: ADH1p-LexA-B42AD-ADHt 2μHIS3 AmpR.

The FUS3 open reading frame was cloned from yeast genomic DNA using:

(SEQ ID NO:3) Primer3: GATCGGATCCATGCCAAAGAGAATTGTATACAATATATC (SEQ IDNO:4) Primer4: ACGCGTCGACTAACTAAATATTTCGTTCCAAATGAGTTTC

The resulting PCR product was digested with BamHI and SalI and ligatedinto Cp5650 that was digested with BamHI and SalI enzymes to generateCp5746: ADH1p-LexA-B42AD-FUS3-ADHt 2μ HIS3 AmpR. Cp5746 was thendigested with EcoRI and Pst I and the 1.3 kb fragment was isolated andligated into Cp5545 that was digested with EcoRI and Pst I enzymes togenerate Cp5766: ADHp-LexA-B42AD-FUS3-ADHt TRP1 CEN6 ARSH4 AmpR.

Cp5545 (ADHp-LexA-MCS-ADHt TRP1 CEN6 ARSH4 AmpR) was generated bycloning the 203–2685 base pair fragment from pLexA (ClontechLaboratories, Inc.) into pRS414 that was digested with EcoRI and SpeI.Although the construct contains an ADH1p, this promoter can be replacedby other promoters that function in yeast well known to the skilledartisan, examples of other promoters include, but are not limited to,PGK, Gal, Cup1, Fus3, and Ste12.

The LBF construct was used to investigate stimulation of the pheromoneresponse pathway using yeast and mammalian heterologous G-proteincoupled receptors. Table 1 shows G-protein coupled receptors that weretested with the LBF construct.

LacZ Assay of Ste2

CY1638 (MATa far 1Δ1442 tbt1-1 fus1-HIS3 trp1 ura3 leu2 his3 suc2) wastransformed with pLexAop(x8)-lacZ (Clontech Laboratories, Ltd.) andCp5766. Transformants were streaked out for single colony purification.A single colony was patched and cells from the patch were frozen down asCY16117. Cells were grown overnight in media lacking uracil andtryptophan. The optical density at 600 nm was determined and thecultures were diluted in fresh media to a final OD₆₀₀ of 0.2. Thecultures were then grown for an additional 2 hours and diluted again toan OD₆₀₀ of 0.2. The lacZ enzyme assay was performed in a 96 well plateand each reaction was performed in duplicate in a total volume of 100 μlwith 90 μl of culture and 10 μl of the ligand. LacZ activities weremeasured at the following concentrations of α-factor: 0 pM, 6.0 pm, 30.2pM, 151 pM, 755 pM, 3.78 nM, 18.9 nM, 94.4 nM, 472 nM, 2.36 μM, 11.8 μMand 59 μM.

Following the addition of ligand, the 96 well plates were incubated at30° C. for either 1, 2 or 3 hours. 20 μl of 1:1 mixture of 1 mMFluorescein di-β-D Galactopyranoside (FDG) in 25 mM Pipes, pH 7.2 and 5%Triton X-100 in 250 mM Pipes, pH 7.2 was then added. The reactions wereincubated at 37° C. for 90 minutes before being stopped by the additionof 20 μl 1M Na₂CO₃ to each well. The plates were read on a fluorometerusing an excitation wavelength of 485 nm and an emission wavelength of535 nm. The data was analyzed using GraphPad Prism software and issummarized in Table 1 below.

LacZ Assay of Melatonin 1a Receptor

CY10981 (MATα GPA1⁺ sst2Δ2 far 1Δ1442 tbt1-1 fus1-HIS3 can1ste14::trp1::LYS2 ste3Δ1156 lys2 ura3 leu2 trp1 his3) was transformedwith pLexAop(x8)-lacZ, Cp5766, and Cp1289 to create CY16328; withpLexAop(x8)-lacZ, Cp5766, and Cp2695 to create strain CY16330. CY12946(MATα GPA1-Gai2(5) sst2Δ2 far1Δ1442 tbt1-1 fus1-HIS3 can1ste14::trp1::LYS2 ste3Δ1156 lys2 ura3 leu2 trp1 his3) was transformedwith the same set to create CY16332 and CY16334.

Cells from each strain were grown overnight in media lacking uracil,tryptophan and leucine, at pH 6.8 with 25 mM PIPES. The optical densityof a 1/10 dilution of the overnight cultures was determined at 600 nmand the cultures were diluted in fresh media to a final OD₆₀₀ of 0.2.The cultures were then grown for an additional 1.5 hours and dilutedagain to an OD₆₀₀ of 0.2. The lacZ enzyme assay was performed in a 96well plate and each reaction was performed in duplicate in a totalvolume of 100 μl with 90 μl of culture and 10 μl of ligand. The finalconcentration of DMSO in each well was kept constant at 1%. LacZactivities, in the presence and absence of the melatonin 1a receptor,were measured at the following concentrations of melatonin: 0 pM, 10 pM,100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 860 μM.

Following the addition of ligand, the 96 well plates were incubated at30° C. for either 1, 2 or 3 hours. 20 μl of 1:1 mixture of 1 mMFluorescein di-β-D Galactopyranoside (FDG) in 25 mM Pipes, pH 7.2 and 5%Triton X-100 in 250 mM Pipes, pH 7.2 was then added. The plates wereincubated at 37° C. for 90 minutes before being stopped by adding 20 μlM Na₂CO₃ to each well. The plates were read on a fluorometer using anexcitation wavelength of 485 nm and an emission wavelength of 535 nm.Table 1 (see below) provides a comparison of the readout obtained usingFUS1p-lacZ and the LBF system (plus the p8op-lacZ reporter gene). Thedata demonstrates that the LBF system produced higher lacZ activitiescompared with FUS1p-lacZ, despite the lower fold induction. Time coursecomparison also showed that the LBF assay can produce a readout after ashorter time period of incubation with the ligand compared with theFUS1p-lacZ construct. These results demonstrate that chimerictranscription factors fused to a pheromone sensitive yeast protein canbe used to a provide a rapid high throughput screening method.

TABLE 1 A summary of G-Protein Coupled Receptors that have been testedwith the LBF system in comparison with FUS1p-lacZ: EC₅₀ 95% ConfidenceMaximum Fold Ligand Interval Induction Incubation Fus1p- Fus1p- ReceptorTime, hrs Gα lacZ LBF lacZ LBF Ste2 1 GPA1 600 nM– 810 nM– 22 29  1 μM 1.3 μM 2 580 nM– 400 nM– 123 63 900 nM 700 nM 3 850 nM– 410 nM– 218 36 1.2 μM 640 nM Melatonin 1 GPA1- 143 nM–  6.3 nM– 54 29 receptor 1ai2(5)  3.3 μM  30 nM 2  6.9 nM– 129 pM– 197 14.3  20 nM 242 pM 3  2.7nM–  37 pM– 302 14.5  6.2 nM  70 pM 1 GPA1  3.4 nM–  1.2 nM– 12 8.1  11nM  4.6 nM 2 460 pM–  70 pM– 68 12  1.2 nM 490 pM 3 140 pM–  38 pM– 1018.4 450 pM  1.1 nM Neurotensin 1 GPA1- 4 11.6 receptor q(5) 2  25 nM–35nM  11 nM–15 nM 11 23 3  25 nM–51 nM  10 nM–13 nM 27 24 Adenosinereceptor 2a 1 Gαs 820 pM– 390 pM–6.1 nM 1.5 1.3 D229S  12 nM 2  3 nM–11nM  6.3 nM–18 nM 2.7 2.4 3  4 nM–10 nM  4.5 nM–9.4 nM 2.7 2.1Somatostatin receptor 1 41-i2 1.3 1.8 2 2 150 nM–  37 nM–416 nM 1.7 1.3282 nM   3.5  45 nM–68 nM  34 nM–71 nM 6 2 1 GPA1- 218 nM– 107 nM– 1.52.3 i2(5) 580 nM 395 nM 2 112 nM–  76 nM–166 nM 4.6 7.8 246 nM   3.5  58nM–92 nM  20 nM–33 nM 36 8.9 Corticotropin 2 GPA1 441 nM–  10 nM–93 μM78 12.5 releasing factor  2.6 μM receptor 1 4 673 nM–1 μM  59 nM–1.9 μM62 4.9 2 Gαs 283 nM– 204 nM–1.1 μM 16.8 6.6 D229S  26 μM 4  37 nM–2.8 μM 14 nM–3.8 μM 13.3 6.3

In addition to the alcohol dehydrogenase promoter (ADHp) LBF construct,other constructs were also generated and tested. Three additionalconstructs, shown in Table 2, were found to be functional in the assaysdescribed above, or in similar assays using the GALp-lacZ reporterconstruct.

TABLE 2 Summary of Additional Chimeric Constructs and theirFunctionality Determined from lacZ Assay Cadus plasmid (Cp) NumberConstruct Function 6231 and 6232 ADHp-LexA-Fus3-B42AD no 6261 and 6262FUS3p-LexA-B42AD-Fus3 yes 6295 and 6296 ADHp-Ga14DBD-B42AD-Fus3* yes6323 and 6324 ADHp-Ga14DBD-Gal4ADII-Fus3* yes *The reporter isGALp-lacZ.

In addition to transformation of the chimeric construct, the constructmay also be integrated into the yeast strain. For example, Cp5766 wastransformed into strain CY7284 (MATa leu2 trp1 his3 ura3 ade2URA3::LexAop-lacZ), which has LexAop-lacZ integrated. The lacZ assay wasperformed as described above. After 3 hours of a-factor treatment,β-galactosidase activities increased by 63 fold (data not shown).

Example 2 Production of Mutant Pheromone Sensitive Yeast Proteins

To investigate further the role of chimeric transcription factors andtheir activation by the pheromone response pathway, mutants of the Fus3pheromone sensitive yeast protein were generated using PCR mutagenesis.The effect of the mutation in the LBF chimeric construct wasinvestigated using methods outlined in Example 1. Mutant Fus3 wasgenerated by amino acid substitution at position 180 and/or at position182, or position 42 of the wild type Fus3 and the following mutants inFus3 were generated in the context of ADHp-LexA-B42AD-Fus3 TRP1 CEN6ARSH4 AmpR:

Construction of Fus3 Mutants at Residues 180 and/or 182

The region encoding the first 183 amino acids of Fus3 was amplifiedusing:

Template: Cp5766

(SEQ ID NO:3) Primer3: GATCGGATCCATGCCAAAGAGAATTGTATACAATATATC (SEQ IDNO:5) Primer5: CCACAWMTTCCWCCATGCCGCTTTGCTGACC(degenerate oligo)where W is A or T, and M is A or C

The resulting PCR product was purified, digested with MscI and BamHI,and ligated into Cp5766 that was digested with the same two enzymes.Mutants of Fus3 were identified by sequencing the PCR product usingstandard sequencing techniques and are summarized in Table 3 below.

Construction of Fus3 Mutant at Residue 42:

The FUS3 open reading frame containing mutation at position 42, whichchanges a lysine residue to an arginine was produced using:

Template: pGA1903,

(SEQ ID NO:3) Primer3: GATCGGATCCATGCCAAAGAGAATTGTATACAATATATC (SEQ IDNO:4) Primer4: ACGCGTCGACTAACTAAATATTTCGTTCCAAATGAGTTTC

The resulting PCR product was digested with BamHI and SalI and ligatedinto Cp5650 that was digested with the same enzymes to generate Cp6082:ADH1p-LexA-B42AD-FUS3(K42R)-ADHt 2μ HIS3 AmpR. Cp6082 was subsequentlydigested with BamHI and MscI and the 555 bp fragment was isolated andligated into Cp5766 that was digested with the same enzymes to generateCp6192. Mutation was confirmed by sequencing. Table 3 show mutants ofFus3 generated in the context of ADHp-LexA-B42AD-Fus3 fusion protein.

TABLE 3 Mutants of Fus3 generated in the context of ADHp-LexA-B42AD-Fus3chimeric construct Cadus Plasmid (Cp) Number Mutation in Fus3 6182Thr180Val, Tyr182Asp 6183 Thr180Glu 6184 Thr180Val, Tyr182Val 6185Thr180Glu, Tyr182Asp 6187 Thr180Glu, Tyr182Val 6189 Thr180Val 6192Lys42Arg

pLexAop(x8)-lacZ and one of the above plasmids or Cp5766 weretransformed into yeast strain CY17206 (MATa bar1 trp1-1a leu2-3, 112ura3 ade1 his2) or CY17327 (MATa bar1 trp1-1a leu2-3, 112 ura3 ade1 his2kss1::LEU2 fus3::ura3Δ5044). Transformants were grown overnight in medialacking uracil and tryptophan. The optical density at 600 nm wasdetermined and the cultures were diluted in fresh media to a final OD₆₀₀of 0.2. The cultures were then grown for an additional 3 hours anddiluted again to an OD₆₀₀ of 0.2. The lacZ enzyme assay was performed ina 96 well plate and each reaction was performed in triplicate in a totalvolume of 100 μl with 95 μl of culture and 5 μl of 0.1 mg/ml α-factor.Following the addition of ligand, the 96 well plates were incubated at30° C. for one hour. 20 μl of 1:1 mixture of 1 mM Fluorescein di-β-DGalactopyranoside (FDG) in 25 mM Pipes, pH 7.2 and 5% Triton X-100 in250 mM Pipes, pH 7.2 was then added. The reactions were incubated at 37°C. for 90 minutes before being stopped by adding 20 μl 1M Na₂CO₃ to eachwell. The plates were read on a fluorometer using an excitationwavelength of 485 nm and an emission wavelength of 535 nm. Table 4provides a summary of the effect of the mutation in Fus3 of the LBFchimeric construct.

TABLE 4 A summary of the effect of the mutation in Fus3 of the LBFchimeric construct. Fold Fold pLexAop(x8)-lacZ Induction in Induction inand Mutation in Fus3 CY17206 CY17327 Cp5766 none 7.1 5.5 Cp6182Thr180Val Tyr182Asp 7.5 1.0 Cp6183 Thrl80Glu 8.3 0.97 Cp6184 Thr180ValTyr182Val 6.5 0.97 Cp6185 Thr180Glu Tyr182Asp 4.3 0.98 Cp6187 Thr180GluTyr182Val 6.7 1.0 Cp6189 Thr180Val 10.7 1.0 Cp6192 Lys42Arg 9.9 1.0

All of the above Fus3 mutants in the chimeric construct were functionalwhen transformed into a FUS3⁺ yeast strain, and resulted in activatedtranscription of the lacZ gene upon α-factor treatment. However, whenthe Fus3 mutants were transformed into a fus3 null yeast strain, theFus3 mutants failed to function. These results indicate a requirementfor the presence of wild type Fus3 for transcriptional activation of thelacZ gene. The wild type Fus3 may be present endogenously in the yeastcell, or may be part of the chimeric transcription factor.

Example 3 A Chimeric Fusion Protein that Confers Pheromone DependentHistidine Prototrophy on a Yeast Cell

The present example describes the use of a pheromone responsive yeastprotein operatively linked to a polypeptide that is required for yeastcell viability. A suitable pheromone responsive yeast protein is, forexample, Ste11. The STE11 coding sequence, or a fragment thereofencoding the Ste11 regulatory domain, is fused to a sequence encodingHis3 (imidazoleglycerol-phosphate dehydratase), a protein required forcell viability. This chimeric construct is then inserted into a yeastexpression vector between the ADH1 promoter and the ADH1 terminator; thevector also contains the TRP1 selectable marker, an autonomousreplicating sequence, and the centromere from yeast chromosome VI. Upontransformation into the appropriate yeast strain, (preferably ahistidine auxotrophic strain), the constitutively expressed chimericSte11-His3 fusion protein confers pheromone pathway-dependent histidineprototrophy on the cell.

The STE11 gene encodes a protein kinase of 738 residues and can beobtained using standard molecular biology techniques. Fusion proteinscomprising the Ste11 protein and a protein required for cell viability,e.g., His3, can be prepared as described in Example 1. The Ste11regulatory domain of Ste11 consists of the N-terminal 415 amino acidresidues. Activation of the yeast pheromone pathway causes thephosphorylation of the Ste11 regulatory domain by Ste20, which in turnstimulates Ste11 to phosphorylate the MAP kinase kinase Ste7, leading toMAP kinase (Fus3) activation.

The HIS3 gene encodes imidazoleglycerol-phosphate dehydratase, an enzymeof 219 residues that catalyzes the formation of imidazoleacetal-phosphate from imidazoleglycerolphosphate, an essential step inhistidine biosynthesis. The fusion of Ste11 or of Ste11ΔC (the aminoterminal regulatory domain alone) to the amino terminus of His3 conferspheromone inducibility upon the activity of the His3 enzyme.

The chimeric construct is transformed into an appropriate yeast strain,(preferably a histidine auxotrophic strain). The appropriate yeaststrain contains a mammalian receptor gene in a yeast expression vector,a G-protein appropriate to that receptor, a deletion of the endogenouspheromone receptor gene (STE2 or STE3), and a lesion in the endogenoushis3 gene, in addition to the expression plasmid for the Ste11-His3chimera. A competitive inhibitor of His3, 3-amino-1,2,4-triazole, can beused in the yeast media to titrate the basal (unstimulated) levels ofHis3 activity.

Constitutive expression of the chimeric Ste11-His3 fusion proteinconfers pheromone pathway-dependent histidine prototrophy on the cell.Thus, a histidine auxotrophic strain that is unable to grow on histidineminus media plates, acquires an ability to grow on these plates uponstimulation of the yeast pheromone pathway.

Incorporation by Reference

All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated herein in theirentireties by reference.

Equivalents

Those skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1. A recombinant yeast cell comprising: a mutated Fus3 yeast proteincomprising an amino acid substitution at positions 42, 180 or 182, or atpositions 180 and 182 as compared to wild type Fus3, and wherein saidyeast protein is operatively linked to a chimeric transcription factor;a heterologous receptor selected from the group consisting of melatoninreceptor 1a, galanin receptor 1, neurotensin receptor, adenosinereceptor 2a, somatostatin receptor 2, and corticotropin releasing factorreceptor 1, wherein said receptor is functionally integrated into apheromone response pathway of the yeast cell; and a gene which producesa detectable protein, wherein said yeast protein is responsive to saidyeast pheromone response pathway of the cell; and wherein the activityof said yeast protein is modulated indirectly upon stimulation of saidpathway, and wherein upon modulation of said yeast protein, saidchimeric transcription factor causes a detectable signal to begenerated.
 2. The recombinant yeast cell of claim 1, wherein said geneis an endogenous gene at its natural location in the yeast cell.
 3. Therecombinant yeast cell of claim 1, wherein said mutated Fus3 yeastprotein is derived from said wild type Fus3 yeast protein.
 4. Therecombinant yeast cell of claim 1, wherein said transcription factor isselected from the group consisting of bacterial, viral and eukaryotictranscription factors.
 5. The recombinant yeast cell of claim 1, whereinsaid chimeric transcription factor comprises a protein selected from thegroup consisting of Ade1, Ade2, Ade3, Ade4, Ade5, Ade7, Ade8, Asp3,Arg1, Arg3, Arg4, Arg5, Arg6, Arg8, Aro2, Aro7, Bar1, CAT, Cho1, Cys3,Gal1, Gal7, Gal10, GFP, His1, His3, His4, His5, Hom3, Hom6, Ilv1, Ilv2,Ilv5, Ino1, Ino2, Ino4, lacZ, Leu1, Leu2, Leu4, luciferase, Lys2, Mal,Mel, Met2, Met3, Met4, Met8, Met9, Met14, Met16, Met19, Ole1, Pho5,Pro1, Pro3, Thr1, Thr4, Trp1, Trp2, Trp3, Trp4, Trp5, Ura1, Ura2, Ura3,Ura4, Ura5, Ura10, Cdc25, Cyr1 and Ras, or fragment thereof.
 6. Therecombinant yeast cell of claim 1, wherein said heterologous receptor ismelatonin receptor 1a.
 7. The recombinant yeast cell of claim 1, whereinsaid chimeric transcription factor comprises an enzyme.
 8. Therecombinant yeast cell of claim 7, wherein said enzyme is selected fromthe group consisting of CAT, Gal1, lacZ, Mel, and Pho5.
 9. Therecombinant yeast cell of claim 1, wherein said chimeric transcriptionfactor comprises a protein required for cell viability.
 10. Therecombinant yeast cell of claim 9, wherein said protein required forcell viability is selected from the group consisting of Gal1, His3,Leu2, Mel, Ura3, Cdc25, Cyr1 and Ras.
 11. The recombinant yeast cell ofclaim 1, wherein said transcription factor comprises an indicatormolecule.
 12. The recombinant yeast cell of claim 11, wherein saidindicator molecule is GFP.
 13. The recombinant yeast cell of claim 1further comprising a promoter operatively linked to said gene, such thatupon modulation of the activity of said yeast protein, the activity ofsaid chimeric transcription factor is stimulated, thereby activatingtranscription of said gene that produces said detectable protein. 14.The recombinant yeast cell of claim 13, wherein said promoter comprisesthe minimal Gal promoter and LexA operators, said gene is lacZ, and saiddetectable protein is β-galactosidase.
 15. The recombinant yeast cell ofclaim 1, wherein the activity of said mutated Fus3 yeast protein ismodulated by wild type Fus3, and wherein the activity of said wild typeFus3 is directly modulated upon stimulation of said pheromone responsepathway.
 16. The recombinant yeast cell of claim 15, wherein saidchimeric transcription factor comprises a LexA DNA binding domainoperatively linked to a B42 transcriptional activation domain.
 17. Therecombinant yeast cell of claim 15, wherein said chimeric transcriptionfactor comprises a Gal4 DNA binding domain operatively linked to a B42transcriptional activation domain.
 18. The recombinant yeast cell ofclaim 15, wherein said chimeric transcription factor comprises a Gal4DNA binding domain operatively linked to a VP16 transcriptionalactivation domain.
 19. The recombinant yeast cell of claim 15, whereinsaid chimeric transcription factor comprises a Gal4 DNA binding domainoperatively linked to a Gal4 transcriptional activation domain II. 20.The recombinant yeast cell of claim 1, wherein said transcription factoris a yeast transcription factor.
 21. The recombinant yeast cell of claim20, wherein said transcription factor is selected from the groupconsisting of Ste12, Gal4, Pho4, Gcn4, Hap1, Adr1, Ace2, Cup2, Swi5 andBas1.
 22. The recombinant yeast cell of claim 21, wherein saidtranscription factor is Ste12.
 23. The recombinant yeast cell of claim21, wherein said transcription factor is Gal4.
 24. The recombinant yeastcell of claim 21, wherein said transcription factor is Pho4.
 25. Therecombinant yeast cell of claim 1, wherein said chimeric transcriptionfactor comprises a DNA binding domain operatively linked to atranscriptional activation domain.
 26. The recombinant yeast cell ofclaim 25, wherein said DNA binding domain comprises a polypeptidesequence derived from a polypeptide selected from the group consistingof LexA, Gal4, Adr1, Ace2, Cup2, Bas1, Gcn4, Swi5, Pho4, Hap1 and LacI,and wherein said transcriptional activation domain comprises apolypeptide sequence derived from a polypeptide selected from the groupconsisting of B42, Gal4, Adr1, Ace2, Cup2, Bas1, Gcn4, Swi5, Pho4, Hap1,VP16, and Ste12.
 27. The recombinant yeast cell of claim 26, whereinsaid DNA binding domain comprises a polypeptide sequence derived fromLexA.
 28. The recombinant yeast cell of claim 26, wherein said DNAbinding domain comprises a polypeptide sequence derived from Gal4. 29.The recombinant yeast cell of claim 26, wherein said DNA binding domaincomprises a polypeptide sequence derived from Pho4.
 30. The recombinantyeast cell of claim 26, wherein said transcriptional activation domaincomprises a polypeptide sequence derived from B42.
 31. The recombinantyeast cell of claim 26, wherein said transcriptional activation domaincomprises a polypeptide sequence derived from VP16.
 32. The recombinantyeast cell of claim 26, wherein said transcriptional activation domaincomprises a polypeptide sequence derived from Gal4.
 33. The recombinantyeast cell of claim 26, wherein said transcriptional activation domaincomprises a polypeptide sequence derived from Ste12.
 34. The recombinantyeast cell of claim 25, wherein said DNA binding domain andtranscriptional activation domain are derived from the same protein. 35.The recombinant yeast cell of claim 34, wherein said chimerictranscription factor comprises a Gal4 DNA binding domain operativelylinked to a Gal4 transcriptional activation domain II.
 36. Therecombinant yeast cell of claim 25, wherein said DNA binding domain andtranscriptional activation domain are derived from different proteins.37. The recombinant yeast cell of claim 36, wherein said chimerictranscription factor comprises a LexA DNA binding domain operativelylinked to a B42 transcriptional activation domain.
 38. The recombinantyeast cell of claim 36, wherein said chimeric transcription factorcomprises a Gal4 DNA binding domain operatively linked to a B42transcriptional activation domain.
 39. The recombinant yeast cell ofclaim 36, wherein said chimeric transcription factor comprises a Gal4DNA binding domain operatively linked to a VP16 transcriptionalactivation domain.
 40. The recombinant yeast cell of claim 1, whereinsaid heterologous receptor is expressed in the membrane of said yeastcell.
 41. The recombinant yeast cell of claim 40, further comprising aheterologous test polypeptide, wherein said heterologous testpolypeptide is transported to a location allowing interaction with theextracellular region of said heterologous receptor, and wherein saidheterologous test polypeptide is expressed at a sufficient level suchthat modulation of the signal transduction activity of the receptor bythe heterologous test polypeptide alters said detectable signal.
 42. Therecombinant yeast cell of claim 41, wherein said heterologous testpolypeptide includes a signal sequence that facilitates transport of thepolypeptide to a location allowing interaction with the extracellularregion of the receptor.
 43. The recombinant yeast cell of claim 1,further comprising a promoter, wherein transcription of said gene iscontrolled by said promoter.
 44. The recombinant yeast cell of claim 43,wherein said promoter is selected from the group consisting of Gal1,Gal10, Mel and LexA operator.
 45. The recombinant yeast cell of claim43, wherein upon modulation of the activity of said yeast protein, saidyeast protein activates transcription of said gene that produces saiddetectable protein.
 46. The recombinant yeast cell of claim 45, whereinsaid detectable protein is selected from the group consisting ofα-galactosidase, β-galactosidase, alkaline phosphatase, horseradishperoxidase, exoglucanase, luciferase, Bar1, Pho5 acid phosphatase, greenfluorescent protein, chitinase, and chloramphenicol acetyl transferase.47. The recombinant yeast cell of claim 45, wherein said gene isselected from the group consisting of ADE1, ADE2, ADE3, ADE4, ADE5,ADE7, ADE8, ASP3, ARG1, ARG3, ARG4, ARG5, ARG6, ARG8, ARO2, ARO7, BAR1,CAT, CHO1, CYS3, GAL1, GAL7, GAL10, GFP, HIS1, HIS3, HIS4, HIS5, HOM3,HOM6, ILV1, ILV2, ILV5, INO1, INO2, INO4, lacZ, LEU1, LEU2, LEU4,luciferase, LYS2, MAL, MEL, MET2, MET3, MET4, MET8, MET9, MET14, MET16,MET19, OLE1, PHO5, PRO1, PRO3, THR1, THR4, TRP1, TRP2, TRP3, TRP4, TRP5,URA1, URA2, URA3, URA4, URA5 and URA10.
 48. The recombinant yeast cellof claim 47, wherein said gene is selected from the group consisting ofCAT, GAL1, GAL7, GAL10, GFP, HIS3, lacZ, luciferase, LEU2, MEL, PHO5,and URA3.
 49. A recombinant yeast cell comprising: a mutated Fus3 yeastprotein comprising an amino acid substitution at positions 42, 180 or182, or at positions 180 and 182 as compared to wild type Fus3, whereinsaid amino acid substitution is selected from the group consisting ofLys42Arg, Thr180Val, Thr180Glu, Tyr182Val and Tyr182Asp and wherein saidyeast protein is operatively linked to a chimeric transcription factor;a heterologous receptor selected from the group consisting of melatoninreceptor 1a, galanin receptor 1, neurotensin receptor, adenosinereceptor 2a, somatostatin receptor 2, and corticotropin releasing factorreceptor 1, wherein said receptor is functionally integrated into apheromone response pathway of the yeast cell; and a gene which producesa detectable protein, wherein said yeast protein is responsive to saidyeast pheromone response pathway of the cell; and wherein the activityof said yeast protein is modulated indirectly upon stimulation of saidpathway, and wherein upon modulation of said yeast protein, saidchimeric transcription factor causes a detectable signal to begenerated.